Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine

ABSTRACT

One aspect of the present invention relates to a double-stranded oligonucleotide comprising at least one ligand. In certain embodiments, a ligand is bound to only one of the two oligonucleotide strands comprising the double-stranded oligonucleotide. In certain embodiments, both of the oligonucleotide strands of the double-stranded oligonucleotide independently comprise a bound ligand. In certain embodiments, the oligonucleotide strands comprise at least one modified sugar moiety. In certain embodiments, a phosphate linkage in one or both of the strands of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. Another aspect of the present invention relates to a single-stranded oligonucleotide comprising at least one ligand. In certain embodiments, the oligonucleotide comprises at least one modified sugar moiety. In certain embodiments, a phosphate linkage of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. The ligand improves the pharmacokinetic properties of the oligonucleotide.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/566,710, filed Apr. 30, 2004; and U.S.Provisional Patent Application Ser. No. 60/620,276, filed Oct. 20, 2004;the contents of both of them are incorporated by reference.

BACKGROUND OF THE INVENTION

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation. Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

siRNA

RNA interference (RNAi) is an evolutionarily conserved gene-silencingmechanism, originally discovered in studies of the nematodeCaenorhabditis elegans (Lee et al, Cell 75:843 (1993); Reinhart et al.,Nature 403:901 (2000)). It is triggered by introducing dsRNA into cellsexpressing the appropriate molecular machinery, which then degrades thecorresponding endogenous mRNA. The mechanism involves conversion ofdsRNA into short RNAs that direct ribonucleases to homologous mRNAtargets (summarized, Ruvkun, Science 2294:797 (2001)). This process isrelated to normal defense against viruses and the mobilization oftransposons.

Double-stranded ribonucleic acids (dsRNAs) are naturally rare and havebeen found only in certain microorganisms, such as yeasts or viruses.Recent reports indicate that dsRNAs are involved in phenomena ofregulation of expression, as well as in the initiation of the synthesisof interferon by cells (Declerq et al., Meth. Enzymol. 78:291 (1981);Wu-Li, Biol. Chem. 265:5470 (1990)). In addition, dsRNA has beenreported to have anti-proliferative properties, which makes it possiblealso to envisage therapeutic applications (Aubel et al., Proc. Natl.Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNA has beenshown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci.USA, 62:357-361 (1969)), to be active in the treatment of leukemic mice(Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)); andto inhibit chemically-induced tumorigenesis in mouse skin (Gelboin etal., Science 167:205-207 (1970)).

Treatment with dsRNA has become an important method for analyzing genefunctions in invertebrate organisms. For example, Dzitoveva et al.showed, that RNAi can be induced in adult fruit flies by injecting dsRNAinto the abdomen of anesthetized Drosophila, and that this method canalso target genes expressed in the central nervous system (Mol.Psychiatry 6(6):665-670 (2001)). Both transgenes and endogenous geneswere successfully silenced in adult Drosophila by intra-abdominalinjection of their respective dsRNA. Moreover, Elbashir et al., providedevidence that the direction of dsRNA processing determines whether senseor antisense target RNA can be cleaved by a small interfering RNA(siRNA)-protein complex (Genes Dev. 15(2): 188-200 (2001)).

Two recent reports reveal that RNAi provides a rapid method to test thefunction of genes in the nematode Caenorhabditis elegans; and most ofthe genes on C. elegans chromosome I and III have now been tested forRNAi phenotypes (Barstead, Curr. Opin. Chem. Biol. 5(1):63-66 (2001);Tavernarakis, Nat. Genet. 24(2):180-183 (2000); Zamore, Nat. Struct.Biol. 8(9):746-750 (2001).). When used as a rapid approach to obtainloss-of-function information, RNAi was used to analyze a random set ofovarian transcripts and has identified 81 genes with essential roles inC. elegans embryogenesis (Piano et al., Curr. Biol. 10(24):1619-1622(2000). RNAi has also been used to disrupt the pupal hemocyte protein ofSarcophaga (Nishikawa et al., Eur. J. Biochem. 268(20):5295-5299(2001)).

Like RNAi in invertebrate animals, post-transcriptional gene-silencing(PTGS) in plants is an RNA-degradation mechanism. In plants, this canoccur at both the transcriptional and the post-transcriptional levels;however, in invertebrates only post-transcriptional RNAi has beenreported to date (Bernstein et al., Nature 409(6818):295-296 (2001).Indeed, both involve double-stranded RNA (dsRNA), spread within theorganism from a localized initiating area, to correlate with theaccumulation of small interfering RNA (siRNA) and require putativeRNA-dependent RNA polymerases, RNA helicases and proteins of unknownfunctions containing PAZ and Piwi domains.

Some differences are evident between RNAi and PTGS were reported byVaucheret et al., J. Cell Sci. 114(Pt 17):3083-3091 (2001). First, PTGSin plants requires at least two genes—SGS3 (which encodes a protein ofunknown function containing a coil-coiled domain) and METI (whichencodes a DNA-methyltransferase)—that are absent in C. elegans, and thusare not required for RNAi. Second, all of the Arabidopsis mutants thatexhibit impaired PTGS are hyper-susceptible to infection by thecucumovirus CMV, indicating that PTGS participates in a mechanism forplant resistance to viruses. RNAi-mediated oncogene silencing has alsobeen reported to confer resistance to crown gall tumorigenesis (Escobaret al., Proc. Natl. Acad. Sci. USA, 98(23):13437-13442 (2001)).

RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multicomponent nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.Hammond et al. (Science 293(5532):1146-1150 (August 2001)) reportedbiochemical purification of the RNAi effector nuclease from culturedDrosophila cells, and protein microsequencing of a ribonucleoproteincomplex of the active fraction showed that one constituent of thiscomplex is a member of the Argonaute family of proteins, which areessential for gene silencing in Caenorhabditis elegans, Neurospora, andArabidopsis. This observation suggests links between the geneticanalysis of RNAi from diverse organisms and the biochemical model ofRNAi that is emerging from Drosophila in vitro systems.

Svoboda et al. reported in Development 127(19):4147-4156 (2000) thatRNAi provides a suitable and robust approach to study the function ofdormant maternal mRNAs in mouse oocytes. Mos (originally known as c-mos)and tissue plasminogen activator mRNAs are dormant maternal mRNAs arerecruited during oocyte maturation, and translation of Mos mRNA resultsin the activation of MAP kinase. The dsRNA directed towards Mos or TPAmRNAs in mouse oocytes specifically reduced the targeted mRNA in both atime- and concentration-dependent manner, and inhibited the appearanceof MAP kinase activity. See also, Svoboda et al. Biochem. Biophys. Res.Commun. 287(5):1099-1104 (2001).

Despite the advances in interference RNA technology, the need exists forsiRNA conjugates having improved pharmacologic properties. Inparticular, the oligonucleotide sequences have poor serum solubility,poor cellular distribution and uptake, and are rapidly excreted throughthe kidneys. It is known that oligonucleotides bearing the nativephospodiester (P═O) backbone are susceptable to nuclease-mediateddegradation. See L. L. Cummins et al. Nucleic Acids Res. 1995, 23, 2019.The stability of oligonucleotides has been increased by converting theP═O linkages to P═S linkages which are less susceptible to degradationby nucleases in vivo. Alternatively, the phosphate group can beconverted to a phosphoramidate which is less prone to enzymaticdegradation than the native phosphate. See Uhlmann, E.; Peyman, A. Chem.Rev. 1990, 90, 544. Modifications to the sugar groups of theoligonucleotide can confer stability to enzymatic degradation. Forexample, oligonucleotides comprising ribonucleic acids are less prone tonucleolytic degradation if the 2′-OH group of the sugar is converted toa methoxyethoxy group. See M. Manoharan ChemBioChem. 2002, 3, 1257 andreferences cited therein.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

Some progress has been made on increasing the cellular uptake ofsingle-stranded oligonucleotides, including increasing the membranepermeability via conjugates and cellular delivery of oligonucleotides.In U.S. Pat. No. 6,656,730, M. Manoharan describes compositions in whicha ligand that binds serum, vascular, or cellular proteins may beattached via an optional linking moiety to one or more sites on anoligonucleotide. These sites include one or more of, but are not limitedto, the 2′-position, 3′-position, 5′-position, the internucleotidelinkage, and a nucleobase atom of any nucleotide residue.

Antisense RNA

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to mRNA or DNA such that the normal, essentialfunctions, such as protein synthesis, of these intracellular nucleicacids are disrupted. Hybridization is the sequence-specific hydrogenbonding via Watson-Crick base pairs of oligonucleotides to RNA orsingle-stranded DNA. Such base pairs are said to be complementary to oneanother.

The naturally-occurring events that provide the disruption of thenucleic acid function, discussed by Cohen (Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., 1989, Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest,describes the terminating event in which the oligonucleotide inhibitorbinds to the target nucleic acid and thus prevents, by simple sterichindrance, the binding of essential proteins, most often ribosomes, tothe nucleic acid. Methyl phosphonate oligonucleotides (Miller et al.(1987) Anti-Cancer Drug Design, 2:117-128), and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

Another means by which antisense oligonucleotides disrupt nucleic acidfunction is by hybridization to a target mRNA, followed by enzymaticcleavage of the targeted RNA by intracellular RNase H. A2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analoghybridizes with the targeted RNA and this duplex activates the RNase Henzyme to cleave the RNA strand, thus destroying the normal function ofthe RNA. Phosphorothioate oligonucleotides are the most prominentexample of an antisense agent that operates by this type of antisenseterminating event.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs as antisense agents fordiagnostics, research applications and potential therapeutic purposes.One of the major hurdles that has only partially been overcome in vivois efficient cellular uptake which is severely hampered by the rapiddegradation and excretion of oligonucleotides. The generally acceptedprocess of cellular uptake is by receptor-mediated endocytosis which isdependent on the temperature and concentration of the oligonucleotidesin serum and extra vascular fluids.

Efforts aimed at improving the transmembrane delivery of nucleic acidsand oligonucleotides have utilized protein carriers, antibody carriers,liposomal delivery systems, electroporation, direct injection, cellfusion, viral vectors, and calcium phosphate-mediated transformation.However, many of these techniques are limited by the types of cells inwhich transmembrane transport is enabled and by the conditions neededfor achieving such transport. An alternative that is particularlyattractive for transmembrane delivery of oligonucleotides ismodification of the physicochemical properties of the oligonucleotide.

Micro-RNA

Micro-RNAs are a large group of small RNAs produced naturally inorganisms, at least some of which regulate the expression of targetgenes. Micro-RNAs are formed from an approximately 70 nucleotidesingle-stranded hairpin precursor transcript by Dicer. V. Ambros et al.Current Biology 2003, 13, 807. In many instances, the micro-RNA istranscribed from a portion of the DNA sequence that previously had noknown function. Micro-RNAs are not translated into proteins, rather theybind to specific messenger RNAs blocking translation. It is thought thatmicro-RNAs base-pair imprecisely with their targets to inhibittranslation. Initially discovered members of the micro-RNA family arelet-7 and lin-4. The let-7 gene encodes a small, highly conserved RNAspecies that regulates the expression of endogenous protein-coding genesduring worm development. The active RNA species is transcribed initiallyas an ˜70 nt precursor, which is post-transcriptionally processed into amature ˜21 nt form. Both let-7 and lin-4 are transcribed as hairpin RNAprecursors which are processed to their mature forms by Dicer enzyme(Lagos-Quintana et al, 2001).

The need exists for oligonucleotide conjugates having improvedpharmacologic properties. In particular, natural oligonucleotidesequences are known to be rapidly degraded and excreted in vivo. Theligand-conjugated oligonucleotide compounds of the invention aresignificantly more stable than natural oligonucleotides, allowing forincreased concentrations of the oligonucleotide in the serum.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a double-strandedoligonucleotide comprising at least one ligand. In certain embodiments,a ligand is bound to only one of the two oligonucleotide strandscomprising the double-stranded oligonucleotide. In certain embodiments,both of the oligonucleotide strands of the double-strandedoligonucleotide independently comprise a bound ligand. In certainembodiments, the oligonucleotide strands comprise at least one modifiedsugar moiety. In certain embodiments, a phosphate linkage in one or bothof the strands of the oligonucleotide has been replaced with aphosphorothioate or phosphorodithioate linkage. In a preferredembodiment, the ligand is cholesterol or 5β-cholanic acid.

Another aspect of the present invention relates to a single-strandedoligonucleotide comprising at least one ligand. In certain embodiments,the oligonucleotide comprises at least one modified sugar moiety. Incertain embodiments, a phosphate linkage of the oligonucleotide has beenreplaced with a phosphorothioate or phosphorodithioate linkage. In apreferred embodiment, the ligand is cholesterol or 5β-cholanic acid. Theligand improves the pharmacokinetic properties of the oligonucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a procedure for solid-phase oligonucleotide synthesis.

FIG. 2 depicts a general procedure for preparing nucleoside-ligandconjugates containing uridine analogues. Note: (i) See Examples (ii)TBDMS-Cl, AgNO₃, Py/THF or TBDMS-Cl, imidazole/Py (iii) Phosphitylation:For Y=Me (or Et): N,N-diisopropylamino, methyl(or ethyl)phosphonamidicchloride {[(CH₃)₂CH]₂N—P(Cl)—CH₃}, DIEA/dichloromethane; For Y=OCH₃:chloro N,N-diisopropyl methoxy phosphoramidite{[(CH₃)₂CH]₂N—P(Cl)—OCH₃}, DIEA/dichloromethane; For Y=OCH₂CH₂CN:N,N-diisopropylamino β-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/dichloromethane or2-cyanoethyl-N,N,N,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/acetonitrile; (iv) (a) succinicanhybride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P, aminoalkylsolid support.

FIG. 3 depicts a general procedure for preparing nucleoside-ligandconjugates containing cytidine analogues. Note: (i) (a) TEA, triazole,POCl₃/MeCN, −10° C.; (b) NH₃; (c) benzoic anhybride/Py (ii) TBDMS-Cl,AgNO₃, Py/THF or TBDMS-Cl, imidazole/Py (iii) Phosphitylation: For Y=Me(or Et): N,N-diisopropylamino, methyl(or ethyl)phosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—CH₃}, DIEA/Dichloromethane; For Y=OCH₃: chloroN,N-diisopropylmethoxyphosphoramidite {[(CH₃)₂CH]₂N—P(Cl)—OCH₃},DIEA/Dichloromethane; For Y=OCH₂CH₂CN: N,N-diisopropylaminoβ-cyanoethylphosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN},DIEA/dichloromethane or 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane,tetrazole (or tetrazolediisopropylammonium salt)/acetonitrile; (iv) (a)succinic anhybride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P,aminoalkyl solid support.

FIG. 4 depicts a general procedure for preparing nucleoside-ligandconjugates wherein the ligand is a carbohydrate. Note: (i) Compound A orB or C, DCC, DMAP, N-hydroxysuccinimide, followed by addition of 1-5 andTEA (ii) TBDMS-Cl, AgNO₃, Py/THF or TBDMS-Cl, Imidazole/Py (iii)Phophitylation: For Y=Me (or Et): N,N-Diisopropylamino, methyl(orethyl)phosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—CH₃},DIEA/dichloromethane; For Y=OCH₃:chloro-N,N-diisopropylmethoxyphosphoramidite {[(CH₃)₂CH]₂N—P(Cl)—OCH₃},DIEA/dichloromethane; For Y=OCH₂CH₂CN: N,N-diisopropylaminoβ-cyanoethylphosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN},DIEA/dichloromethane or 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane,tetrazole (or tetrazolediisopropylammonium salt)/acetonitrile; (iv) (a)succinic anhydride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P,aminoalkyl solid support.

FIG. 5 depicts a general procedure for preparing nucleoside-ligandconjugates wherein the ligand is a biotin analog. Note: (i) (a) BiotinNHS ester TEA; (b) TMSCl and then 4-tert-buytlbenzoyl chloride, DMAP/Py(ii) TBDMSCl, AgNO₃, Py/THF or TBDMS-Cl, imidazole/Py (iii)Phophitylation: For Y=Me (or Et): N,N-diisopropylamino, methyl(orethyl)phosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—CH₃},DIEA/dichloromethane; For Y=OCH₃: chloro N,N-diisopropyl methoxyphosphoramidite {[(CH₃)₂CH]₂N—P(Cl)—OCH₃}, DIEA/dichloromethane; ForY=OCH₂CH₂CN: N,N-diisopropylamino β-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/dichloromethane or2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/acetonitrile; (iv) (a) succinicanhydride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P, aminoalkylsolid support

FIG. 6 depicts a general procedure for preparing nucleoside-ligandconjugates. Note: (i) (a) compound 161, DCC, DMAP, N-hydroxysuccinimide,followed by addition of 1-5 and TEA, or (b) compound 162,N,N-disuccinimidyl carbonate, TEA followed by addition of 1-5 (ii)TBDMS-Cl, AgNO₃, Py/THF or TBDMS-Cl, imidazole/Py (iii) Phophitylation:For Y=Me (or Et): N,N-diisopropylamino, methyl(or ethyl)phosphonamidicchloride {[(CH₃)₂CH]₂N—P(Cl)—CH₃}, DIEA/dichloromethane; For Y=OCH₃:chloro-N,N-diisopropyl methoxy phosphoramidite{[(CH₃)₂CH]₂N—P(Cl)—OCH₃}, DIEA/dichloromethane; For Y=OCH₂CH₂CN:N,N-diisopropylamino β-cyanoethylphosphonamidic chloride{[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN}, DIEA/dichloromethane or2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane, tetrazole (ortetrazolediisopropylammonium salt)/acetonitrile; (iv) (a) succinicanhydride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P, aminoalkylsolid support.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ligand-conjugated oliognucleotidecompounds having improved pharmacokinetic properties and methods fortheir preparation. The oligonucleotides of the invention includesingle-stranded and double-stranded oligonucleotides. Conjugatedoligonucleotide agents can modify gene expression, either inhibiting orup-regulating, by targeting and binding to a nucleic acid, e.g., apre-mRNA, an mRNA, a microRNA (miRNA), a mi-RNA precursor (pre-miRNA),or DNA, or to a protein. Oligonucleotide agents of the invention includemodified siRNA, miRNA, antisense RNA, decoy RNA, DNA, and aptamers. Suchcompounds are prepared by covalently attaching a ligand to anoliognucleotide. The ligand improves the pharmacologic properties of theoliognucleotide because it binds reversibly to one or more serum,vascular or cellular proteins. This reversible binding is expected todecrease urinary excretion, increase serum half-life and greatlyincrease the distribution of oligomeric compounds thus conjugated.

The therapeutic effect of an oliognucleotide is realized when itinteracts with a specific cellular nucleic acid and effectively negatesits function. A preferred target is DNA or mRNA encoding a protein thatis responsible for a disease state. The overall effect of suchinterference with mRNA function is modulation of the expression of aprotein, wherein “modulation” means either an increase (stimulation) ora decrease (inhibition) in the expression of the protein. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression. Nevertheless, the ultimate goal is to regulate theamount of such a protein.

To reach a target nucleic acid after administration, an oliognucleotideshould be able to overcome inherent factors such as rapid degradation inserum, short half-life in serum and rapid filtration by the kidneys withsubsequent excretion in the urine. Oliognucleotides that overcome theseinherent factors have increased serum half-life, distribution, cellularuptake and hence improved efficacy.

These enhanced pharmacokinetic parameters have been shown for selecteddrug molecules that bind plasma proteins (Olson and Christ, AnnualReports in Medicinal Chemistry, 1996, 31:327). Two proteins that havebeen studied more than most are human serum albumin (HSA) and α-1-acidglycoprotein. HSA binds a variety of endogenous and exogenous ligandswith association constants typically in the range of 10⁴ to 10⁶ M⁻¹.Association constants for ligands with α-1-acid glycoprotein are similarto those for HSA.

In a preferred embodiment of the invention the protein targeted by theligand-conjugated oliognucleotide is a serum protein. It is preferredthat the serum protein targeted by a conjugated oligomeric compound isan immunoglobulin (an antibody). Preferred immunoglobulins areimmunoglobulin G and immunoglobulin M. Immunoglobulins are known toappear in blood serum and tissues of vertebrate animals.

In another embodiment of the invention the serum protein targeted by theligand-conjugated oliognucleotide is a lipoprotein. Lipoproteins areblood proteins having molecular weights generally above 20,000 thatcarry lipids and are recognized by specific cell-surface receptors. Theassociation with lipoproteins in the serum will initially increasepharmacokinetic parameters such as half-life and distribution. Asecondary consideration is the ability of lipoproteins to enhancecellular uptake via receptor-mediated endocytosis.

In yet another embodiment the serum protein targeted by theligand-conjugated oliognucleotide compound is α-2-macroglobulin. In yeta further embodiment the serum protein targeted by a ligand conjugatedoligomeric compound is α-1-glycoprotein.

At least for therapeutic purposes, oliognucleotide compounds should havea degree of stability in serum to allow distribution and cellularuptake. The prolonged maintenance of therapeutic levels of antisenseagents in serum will have a significant effect on the distribution andcellular uptake and unlike conjugate groups that target specificcellular receptors, the increased serum stability will effect all cells.

In the context of this invention, siRNA comprises double-strandedoligonucleotides, wherein the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid or deoxyribonucleic acid. Thisterm includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas modified oligonucleotides having non-naturally-occurring portionswhich function similarly. Such modified or substituted oligonucleotidesare often preferred over native forms because of desirable propertiessuch as, for example, enhanced cellular uptake, enhanced binding totarget and increased stability in the presence of nucleases. Theoligonucleotides of the present invention preferably comprise from about5 to about 50 nucleosides. It is more preferred that sucholigonucleotides comprise from about 8 to about 30 nucleosides, with 15to 25 nucleosides being particularly preferred.

An oligonucleotide is a polymer of repeating units generically known asnucleotides or nucleosides. An unmodified (naturally occurring)nucleotide has three components: (1) a nitrogenous base linked by one ofits nitrogen atoms to (2) a 5-carbon cyclic sugar and (3) a phosphate,esterified to carbon 5 of the sugar. When incorporated into anoligonucleotide chain, the phosphate of a first nucleotide is alsoesterified to carbon 3 of the sugar of a second, adjacent nucleotide.The “backbone” of an unmodified oligonucleotide consists of (2) and (3),that is, sugars linked together by phosphodiester linkages between theCS (5′) position of the sugar of a first nucleotide and the C3 (3′)position of a second, adjacent nucleotide. A “nucleoside” is thecombination of (1) a nucleobase and (2) a sugar in the absence of aphosphate moiety (Komberg, DNA Replication, W. H. Freeman & Co., SanFrancisco, 1980, pages 4-7). The backbone of an oligonucleotidepositions a series of bases in a specific order; the writtenrepresentation of this series of bases, which is conventionally writtenin 5′ to 3′ order, is known as a nucleotide sequence.

Oligonucleotides may comprise nucleoside or nucleotide sequencessufficient in identity and number to effect specific hybridization witha particular nucleic acid. Such oligonucleotides which specificallyhybridize to a portion of the sense strand of a gene are commonlydescribed as “antisense.” In the context of the invention,“hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleosides or nucleotides. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. “Complementary,” as used herein, refers to the capacity forprecise pairing between two nucleotides. For example, if a nucleotide ata certain position of an oligonucleotide is capable of hydrogen bondingwith a nucleotide at the same position of a DNA or RNA molecule, thenthe oligonucleotide and the DNA or RNA are considered to becomplementary to each other at that position. The oligonucleotide andthe DNA or RNA are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that an oligonucleotide need notbe 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA to cause adecrease or loss of function, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

The ligand-conjugated oligonucleotides of the invention can be preparedby attaching the ligand to the oligonucleotide through a monomer, e.g.,a chemically modified monomer that is integrated into theoligonucleotide agent. In a preferred embodiment, the coupling is by atether or a linker (or both) as described below, and the complex has theformula represented by:Ligand-[linker]_(optional)-[tether]_(optional)-oligonucleotide agent

While, in most cases, embodiments are described with respect to anoligonucleotide agent including a number of nucleotides, the inventionalso includes monomeric subunits having the structure:Ligand-[linker]_(optional)-[tether]_(optional)-monomer

Methods of making and incorporating the monomers into theoligonucleotide agents and methods of using those agents are included inthe invention. In preferred embodiments, the sugar, e.g., the ribosesugar of one or more of the nucleotides, (e.g., ribonucleotide,deoxynucleotide, or modified nucleotide) subunits of an oligonucleotideagent can be replaced with another moiety, e.g., a non-carbohydratecarrier. In certain instances, the non-carbohydrate is cyclic. Anucleotide subunit in which the sugar of the subunit has been soreplaced is referred to herein as a sugar replacement modificationsubunit (SRMS). This is often referred to as a tether. A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atomsor a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, or sulfur. The cyclic carrier may bea monocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The oligonucleotide agents of the invention include nucleic acidtargeting (NAT) oligonucleotide agents and protein-targeting (PT)oligonucleotide agents. NAT and PT oligonucleotide agents refer tosingle-stranded oligomers or polymers of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or combined (chimeric) modifications of DNAand RNA. This term includes oligonucleotides composed of naturallyoccurring nucleobases, sugars, and covalent internucleoside (backbone)linkages as well as oligonucleotides having non-naturally-occurringportions that function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofdesirable properties such as enhanced cellular uptake, enhanced affinityfor nucleic acid target, and/or increased stability in the presence ofnucleases. NATs designed to bind to specific RNA or DNA targets havesubstantial complementarity, e.g., at least 70, 80, 90, or 100%complementary, with at least 10, 20, or 30 or more bases of a targetnucleic acid, and include antisense RNAs, miRNAs, and other non-duplexstructures which can modulate expression. Other NAT oligonucleotideagents include external guide sequence (EGS) oligonucleotides(oligozymes), DNAzymes, and ribozymes. These NATs may or may not bindvia Watson-Crick complementarity to their targets. PT oligonucleotideagents bind to protein targets, preferably by virtue ofthree-dimensional interactions, and modulate protein activity. Theyinclude decoy RNAs, aptamers, and the like.

The single-stranded oligonucleotide compounds of the inventionpreferably comprise from about 8 to about 50 nucleobases (i.e. fromabout 8 to about 50 linked nucleosides). NAT oligonucleotide agents arepreferably about 15 nucleotides long, or more preferably about 30nucleotides long. PT oligonucleotide agents are preferably about 18nucleotides long, or more preferably about 23 nucleotides long.Particularly preferred compounds are miRNAs and antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases.

While not wishing to be bound by theory, an oligonucleotide agent mayact by one or more of a number of mechanisms, including acleavage-dependent or cleavage-independent mechanism. A cleavage-basedmechanism can be RNAse H dependent and/or can include RISC complexfunction. Cleavage-independent mechanisms include occupancy-basedtranslational arrest, such as is mediated by miRNAs, or binding of theoligonucleotide agent to a protein, as do aptamers. Oligonucleotideagents may also be used to alter the expression of genes by changing thechoice of the splice site in a pre-mRNA. Inhibition of splicing can alsoresult in degradation of the improperly processed message, thusdown-regulating gene expression. Kole and colleagues (Sierakowska, etal. Proc. Natl. Acad. Sci. USA, 1996, 93:12840-12844) showed that2′-O-Me phosphorothioate oligonucleotides could correct aberrantbeta-globin splicing in a cellular system. Fully modified2′-methoxyethyl oligonucleotides and peptide nucleic acids (PNAs) wereable to redirect splicing of IL-5 receptor-α pre-mRNA (Karras et al.,Mol. Pharmacol. 2000, 58:380-387; Karras, et al., Biochemistry 2001,40:7853-7859).

MicroRNAs

The oligonucleotide agents include microRNAs (miRNAs). MicroRNAs aresmall noncoding RNA molecules that are capable of causingpost-transcriptional silencing of specific genes in cells such as by theinhibition of translation or through degradation of the targeted mRNA. AmiRNA can be completely complementary or can have a region ofnoncomplementarity with a target nucleic acid, consequently resulting ina “bulge” at the region of non-complementarity. The region ofnon-complementarity (the bulge) can be flanked by regions of sufficientcomplementarity, preferably complete complementarity to allow duplexformation. Preferably, the regions of complementarity are at least 8 to10 nucleotides long (e.g., 8, 9, or 10 nucleotides long). A miRNA caninhibit gene expression by repressing translation, such as when themicroRNA is not completely complementary to the target nucleic acid, orby causing target RNA degradation, which is believed to occur only whenthe miRNA binds its target with perfect complementarity. The inventionalso includes double-stranded precursors of miRNAs that may or may notform a bulge when bound to their targets.

A miRNA or pre-miRNA can be about 18-100 nucleotides in length, and morepreferably from about 18-80 nucleotides in length. Mature miRNAs canhave a length of about 19-30 nucleotides, preferably about 21-25nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MicroRNAprecursors can have a length of about 70-100 nucleotides and have ahairpin conformation. MicroRNAs can be generated in vivo from pre-miRNAsby enzymes called Dicer and Drosha that specifically process longpre-miRNA into functional miRNA. The microRNAs or precursor miRNAsfeatured in the invention can be synthesized in vivo by a cell-basedsystem or can be chemically synthesized. MicroRNAs can be synthesized toinclude a modification that imparts a desired characteristic. Forexample, the modification can improve stability, hybridizationthermodynamics with a target nucleic acid, targeting to a particulartissue or cell-type, or cell permeability, e.g., by anendocytosis-dependent or -independent mechanism. Modifications can alsoincrease sequence specificity, and consequently decrease off-sitetargeting. Methods of synthesis and chemical modifications are describedin greater detail below.

In particular, an miRNA or a pre-miRNA featured in the invention canhave a chemical modification on a nucleotide in an internal (i.e.,non-terminal) region having noncomplementarity with the target nucleicacid. For example, a modified nucleotide can be incorporated into theregion of a miRNA that forms a bulge. The modification can include aligand attached to the miRNA, e.g., by a linker. The modification can,for example, improve pharmacokinetics or stability of a therapeuticmiRNA, or improve hybridization properties (e.g., hybridizationthermodynamics) of the miRNA to a target nucleic acid. In someembodiments, it is preferred that the orientation of a modification orligand incorporated into or tethered to the bulge region of a miRNA isoriented to occupy the space in the bulge region. This orientationfacilitates the improved hybridization properties or an otherwisedesired characteristic of the miRNA. For example, the modification caninclude a modified base or sugar on the nucleic acid strand or a ligandthat functions as an intercalator. These are preferably located in thebulge. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. Universal bases can also be incorporated into the miRNAs.

In one embodiment, an miRNA or a pre-miRNA can include an aminoglycosideligand, which can cause the miRNA to have improved hybridizationproperties or improved sequence specificity. Exemplary aminoglycosidesinclude glycosylated polylysine; galactosylated polylysine; neomycin B;tobramycin; kanamycin A; and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity.Neo-S-acridine, an acridine analog, has an increased affinity for theHIV Rev-response element (RRE). In some embodiments, the guanidineanalog (the guanidinoglycoside) of an aminoglycoside ligand is tetheredto an oligonucleotide agent. In a guanidinoglycoside, the amine group onthe amino acid is exchanged for a guanidine group. Attachment of aguanidine analog can enhance cell permeability of an oligonucleotideagent.

In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. Preferably, the cleaving group is tethered to the miRNA in amanner such that it is positioned in the bulge region, where it canaccess and cleave the target RNA. The cleaving group can be, forexample, a bleomycin (e.g., bleomycin-A₅, bleomycin-A₂, orbleomycin-B₂), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal ionchelating group. The metal ion chelating group can include, e.g., anLu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a miRNA or a pre-miRNAto promote cleavage of the target RNA, such as at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. The methods and compositions featured inthe invention include miRNAs that inhibit target gene expression by acleavage or non-cleavage dependent mechanism.

A miRNA or a pre-miRNA can be designed and synthesized to include aregion of noncomplementarity (e.g., a region that is 3, 4, 5, or 6nucleotides long) flanked by regions of sufficient complementarity toform a duplex (e.g., regions that are 7, 8, 9, 10, or 11 nucleotideslong). For increased nuclease resistance and/or binding affinity to thetarget, the miRNA sequences can include 2′-O-methyl, 2′-fluorine,2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and/or phosphorothioatelinkages. The inclusion of furanose sugars in the oligonucleotidebackbone can also decrease endonucleolytic cleavage. An miRNA or apre-miRNA can be further modified by including a 3′-cationic group, orby inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. Inanother alternative, the 3′-terminus can be blocked with an aminoalkylgroup, e.g., a 3′-C5-aminoalkyl dT. Other 3′-conjugates can inhibit3′-5′ exonucleolytic cleavage. While not being bound by theory, a3′-conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

In one embodiment, a miRNA or a pre-miRNA includes a modification thatimproves targeting, e.g. a targeting modification described above.Examples of modifications that target miRNA molecules to particular celltypes include carbohydrate sugars such as galactose,N-acetylgalactosamine, mannose; vitamins such as folates; other ligandssuch as RGDs and RGD mimics; and small molecules including naproxen,ibuprofen or other known protein-binding molecules.

A miRNA or a pre-miRNA can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, a miRNA or a pre-miRNA can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between the miRNAor a pre-miRNA and target nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Otherappropriate nucleic acid modifications are described herein.Alternatively, the miRNA or pre-miRNA nucleic acid can be producedbiologically using an expression vector into which a nucleic acid hasbeen subcloned in an antisense orientation, i.e., RNA transcribed fromthe inserted nucleic acid will be of an antisense orientation to atarget nucleic acid of interest.

Antisense Nucleic Acid Sequences

The single-stranded oligonucleotide agents featured in the inventioninclude antisense nucleic acids. An “antisense” nucleic acid includes anucleotide sequence that is complementary to a “sense” nucleic acidencoding a gene expression product, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an RNAsequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, anantisense nucleic acid can form hydrogen bonds with a sense nucleic acidtarget.

Given a coding strand sequence such as the sequence of a sense strand ofa cDNA molecule, antisense nucleic acids can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to a portion of the coding or noncodingregion of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of a pre-mRNA or mRNA, e.g., the 5′ UTR. Anantisense oligonucleotide can be about 10 to 25 nucleotides in length(e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotidesin length). An antisense oligonucleotide can also be complementary to amiRNA or pre-miRNA.

An antisense nucleic acid can be constructed using chemical synthesisand/or enzymatic ligation reactions using procedures known in the art.For example, an antisense nucleic acid can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and target nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Other appropriatenucleic acid modifications are described herein. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation, i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest.

An antisense agent can include ribonucleotides only,deoxyribonucleotides only (e.g., oligodeoxynucleotides), or bothdeoxyribonucleotides and ribonucleotides. For example, an antisenseagent consisting only of ribonucleotides can hybridize to acomplementary RNA, and prevent access of the translation machinery tothe target RNA transcript, thereby preventing protein synthesis. Anantisense molecule including only deoxyribonucleotides, ordeoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked byRNA sequence at the 5′ and 3′ ends of the antisense agent, can hybridizeto a complementary RNA, and the RNA target can be subsequently cleavedby an enzyme such as RNAse H. Degradation of the target RNA preventstranslation. The flanking RNA sequences can include 2′-O-methylatednucleotides, and phosphorothioate linkages, and the internal DNAsequence can include phosphorothioate internucleotide linkages. Theinternal DNA sequence is preferably at least five nucleotides in lengthwhen targeting by RNAse H activity is desired.

For increased nuclease resistance, an antisense agent can be furthermodified by inverting the nucleoside at the 3′-terminus with a 3′-3′linkage. In another alternative, the 3′-terminus can be blocked with anaminoalkyl group. In certain instances, the antisense oligonucleotideagent includes a modification that improves targeting, e.g. a targetingmodification.

Decoy Nucleic Acids

An oligonucleotide agent featured in the invention can be a decoynucleic acid such as decoy RNA. A decoy nucleic acid resembles a naturalnucleic acid, but is modified to inhibit or interrupt the activity ofthe natural nucleic acid. For example, a decoy RNA can mimic the naturalbinding domain for a ligand, and compete with natural binding target forthe binding of a specific ligand. It has been shown that over-expressionof HIV trans-activation response (TAR) RNA can act as a “decoy” andefficiently bind HIV tat protein, thereby preventing it from binding toTAR sequences encoded in the HIV RNA. In one embodiment, a decoy RNAincludes a modification that improves targeting. The chemicalmodifications described above for miRNAs and antisense RNAs, anddescribed elsewhere herein, are also appropriate for use in decoynucleic acids.

Aptamers

Oligonucleotide agents of the invention also include aptamers. Anaptamer binds to a non-nucleic acid ligand, such as a small organicmolecule or protein, e.g., a transcription or translation factor, andsubsequently modifies its activity. An aptamer can fold into a specificstructure that directs the recognition of the targeted binding site onthe non-nucleic acid ligand. An aptamer can contain any of themodifications described herein. In certain instances, the aptamerincludes a modification that improves targeting, e.g., a targetingmodification. The chemical modifications described above for miRNAs andantisense RNAs, and described elsewhere herein, are also appropriate foruse in decoy nucleic acids.

Additional Features of the Oligonucleotides of the Invention

An oligonucleotide agent that is NAT (“nucleic acid targeting”) includesa region of sufficient complementarity to the target gene, and is ofsufficient length in terms of nucleotides, such that the oligonucleotideagent forms a duplex with the target nucleic acid. The oligonucleotideagent can modulate the function of the targeted molecule. For example,when the targeted molecule is an mRNA or pre-mRNA, the NAT can inhibitgene expression; when the target is an miRNA, the NAT will inhibit themiRNA function and will thus up-regulate expression of the mRNAstargeted by the particular miRNA. Alternatively, when the target is aregion of a pre-mRNA that affects splicing, the NAT can alter the choiceof splice site and thus the mRNA sequence; when the NAT functions as anmiRNA, expression of the targeted mRNA is inhibited. For ease ofexposition the term nucleotide or ribonucleotide is sometimes usedherein in reference to one or more monomeric subunits of anoligonucleotide agent. It will be understood that the term“ribonucleotide” or “nucleotide” can, in the case of a modified RNA ornucleotide surrogate, also refer to a modified nucleotide, or surrogatereplacement moiety at one or more positions.

A NAT oligonucleotide agent is, or includes, a region that is at leastpartially, and in some embodiments fully, complementary to the targetRNA. It is not necessary that there be perfect complementarity betweenthe oligonucleotide agent and the target, but the correspondence must besufficient to enable the oligonucleotide agent, or a cleavage productthereof, to modulate (e.g., inhibit) target gene expression.

The oligonucleotide agent will preferably have one or more of thefollowing properties: (1) it will have a 5′ modification that includesone or more phosphate groups or one or more analogs of a phosphategroup; (2) it will, despite modifications even to a very large number ofbases, specifically base pair and form a duplex structure with ahomologous target RNA of sufficient thermodynamic stability to allowmodulation of the activity of the targeted RNA; and (3) it will, despitemodifications even to a very large number, or all of the nucleosides,still have “RNA-like” properties, i.e., it will possess the overallstructural, chemical and physical properties of an RNA molecule, eventhough not exclusively, or even partly, of ribonucleotide-based content.For example, all of the nucleotide sugars can contain a 2′-fluoro groupin place of 2′-hydroxyl group. This deoxyribonucleotide-containing agentcan still be expected to exhibit RNA-like properties. While not wishingto be bound by theory, the electronegative fluorine prefers an axialorientation when attached to the C2′-position of ribose. This spatialpreference of fluorine can force the sugars to adopt a C_(3′)-endopucker. This is the same puckering mode as observed in RNA molecules andgives rise to the RNA-characteristic A-family-type helix. Further, sincefluorine is a good hydrogen bond acceptor, it can participate in thesame hydrogen bonding interactions with water molecules that are knownto stabilize RNA structures. Generally, it is preferred that a modifiedmoiety at the 2′-sugar position will be able to enter intohydrogen-bonding which is more characteristic of the 2′-OH moiety of aribonucleotide than the 2′-H moiety of a deoxyribonucleotide. Apreferred oligonucleotide agent will: exhibit a C_(3′)-endo pucker inall, or at least about 50, 75, 80, 85, 90, or 95% of its sugars; exhibita C_(3′)-endo pucker in a sufficient amount of its sugars that it cangive rise to the RNA-characteristic A-family-type helix; will generallyhave no more than about 20, 10, 5, 4, 3, 2, or 1 sugar which is not aC_(3′)-endo pucker structure. In certain instances, oligonucleotide willexhibit C_(3′)-endo suger pucker and be modified at the 2′-position.Exemplary modifications include 2′-OH, 2′-O-Me, 2′-O-methoxyethyl,2′-O-aminopropyl, 2′-F, 2′-O—CH₂—CO—NHMe, 2′-O—CH₂—CH₂—O—CH₂—CH₂—N(Me)₂,and LNA. In certain instances, regardless of the nature of themodification, and even though the oligonucleotide agent can containdeoxynucleotides or modified deoxynucleotides, it is preferred that DNAmolecules, or any molecule in which more than 50, 60, or 70% of thenucleotides in the molecule are deoxyribonucleotides, or modifieddeoxyribonucleotides which are deoxy at the 2′ position, are excludedfrom the definition of oligonucleotide agent. Some preferred2′-modifications with of sugar moieties exhibiting C2′-endo sugar puckerinclude 2′-H, 2′-Me, 2′-S-Me, 2′-Ethynyl, and 2′-ara-F. Additional sugarmodifications include L-sugars and 2′-5′-linked sugars.

As used herein, “specifically hybridizable” and “complementary” areterms that are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. This nomenclature also applies toinstances when NAT oligonucleotides agents bind to target RNAs. Specificbinding requires a sufficient lack of complementarity to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, or in the case of in vitro assays, underconditions in which the assays are performed. It has been shown that asingle mismatch between targeted and non-targeted sequences aresufficient to provide discrimination for siRNA targeting of an mRNA(Brummelkamp et al., Cancer Cell, 2002, 2:243).

In certain instances, a NAT oligonucleotide agent is “sufficientlycomplementary” to a target RNA, such that the oligonucleotide agentinhibits production of protein encoded by the target mRNA. The targetRNA can be a pre-mRNA, mRNA, or miRNA endogenous to the subject. Inanother embodiment, the oligonucleotide agent is “exactly complementary”(excluding the SRMS containing subunit(s)) to a target RNA, e.g., thetarget RNA and the oligonucleotide agent can anneal to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can include aregion (e.g., of at least about 7 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in some embodiments, theoligonucleotide agent specifically discriminates a single-nucleotidedifference. In this case, the oligonucleotide agent only down-regulatesgene expression if exact complementary is found in the region thesingle-nucleotide difference.

Oligonucleotide agents discussed include otherwise unmodified RNA andDNA as well as RNA and DNA that have been modified. Examples of modifiedRNA and DNA include modificiations to improve efficacy and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The literaturehas referred to rare or unusual, but naturally occurring, RNAs asmodified RNAs. See Limbach et al. Nucleic Acids Res. 1994, 22,2183-2196. Such rare or unusual RNAs, often termed modified RNAs, aretypically the result of a post-transcriptional modification and arewithin the scope of the term unmodified RNA as used herein. Modified RNAas used herein refers to a molecule in which one or more of thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are different from that which occur in nature, preferablydifferent from that which occurs in the human body. While they arereferred to as “modified RNAs” they will of course, because of themodification, include molecules that are not, strictly speaking, RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone.

Sugar Replacement Monomer Subunits (SRMS)

A nucleotide subunit in which the sugar of the subunit has been soreplaced is referred to herein as a sugar replacement modificationsubunit (SRMS). The SRMS includes two “backbone attachment points”(hydroxyl groups), a “tethering attachment point,” and a ligand, whichis connected indirectly to the SRMS via an intervening tether. The SRMSmay be the 5′- or 3′-terminal subunit of the oligonucleotide agent andlocated adjacent to two or more unmodified or modified ribonucleotides.Alternatively, the SRMS may occupy an internal position located adjacentto one or more unmodified or modified ribonucleotides. More than oneSRMS may be present in an oligonucleotide agent. Preferred positions forinclusion of a SRMS tethered to a moiety (e.g., a lipophilic moiety suchas cholesterol) are at the 3′-terminus, the 5′-terminus, or at aninternal position.

Ligands

A wide variety of entities can be tethered to the oligonucleotide agent.A ligand tethered to an oligonucleotide agent can have a favorableeffect on the agent. For example, the ligand can improve stability,hybridization thermodynamics with a target nucleic acid, targeting to aparticular tissue or cell-type, or cell permeability, e.g., by anendocytosis-dependent or -independent mechanism. Ligands and associatedmodifications can also increase sequence specificity and consequentlydecrease off-site targeting. A tethered ligand can include one or moremodified bases or sugars that can function as intercalators. These arepreferably located in an internal region, such as in a bulge of amiRNA/target duplex. The intercalator can be an aromatic group includingpolycyclic aromatics or heterocyclic aromatic groups. A polycyclicintercalator can have stacking capabilities, and can include systemswith 2, 3, or 4 fused rings. Universal bases can be included on aligand.

In one embodiment, the ligand includes a cleaving group that contributesto target gene inhibition by cleavage of the target nucleic acid. Thecleaving group can be a bleomycin (e.g., bleomycin-A5, bleomycin-A2, orbleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), apolyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal-ionchelating group. The metal-ion chelating group can be an Lu(III) orEU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthrolinederivative, a Cu(II) terpyridine, or acridine, which can promote theselective cleavage of target RNA at the site of the bulge by free metalions such as Lu(III). In some instances, a peptide ligand can betethered to a miRNA to promote cleavage of the target RNA. In certaininstances, the cleavage may occur at the bulge region. For example,1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can beconjugated to a peptide, such as via an amino acid derivative, topromote target RNA cleavage.

A tethered ligand can be an aminoglycoside ligand which can cause anoligonucleotide agent to have improved hybridization properties orimproved sequence specificity. Exemplary aminoglycosides includeglycosylated polylysine, galactosylated polylysine, neomycin B,tobramycin, kanamycin A, and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity. Anacridine analog, neo-S-acridine has an increased affinity for the HIVRev-response element (RRE). In some embodiments the guanidine analog(the guanidinoglycoside) of an aminoglycoside ligand is tethered to anoligonucleotide agent. In a guanidinoglycoside, the amine group on theamino acid is exchanged for a guanidine group. Attachment of a guanidineanalog can enhance cell permeability of an oligonucleotide agent. Atethered ligand can be a poly-arginine peptide, peptoid orpeptidomimetic, which can enhance the cellular uptake of anoligonucleotide agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe SRMS carrier. In preferred embodiments, the ligand is attached tothe carrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the SRMS monomer when the SRMS monomeris incorporated into the growing strand. In some embodiments, the ligandmay be incorporated into a “precursor” SRMS after a “precursor” SRMSmonomer has been incorporated into the growing strand. For example, anSRMS monomer having an amino-terminated tether (i.e., having noassociated ligand), or TAP-(CH₂)_(n)NH₂ may be incorporated into agrowing oligonucleotide strand. In a subsequent operation, a ligandhaving an electrophilic group can subsequently be attached to theprecursor SRMS by coupling the electrophilic group of the ligand with aterminal nucleophilic group of the precursor SRMS tether. Representativeelectrophilic groups include pentafluorophenyl esters or an aldehyde.Other electrophilic groups amenable to the present invention can bereadily determined by one of ordinary skill in the art.

Preparation of Oligonucleotides Bearing a Peptide Conjugate

Oligonucleotides bearing peptide conjugates can be prepared usingprocedures analagous to those described below for the preparation ofoligonucleotides bearing ligands. The synthesis and purification ofoligonucleotide peptide conjugates can be performed by establishedmethods. See Trufert et al., Tetrahedron 1996, 52, 3005; and Manoharan,“Oligonucleotide Conjugates in Antisense Technology,” in Antisense DrugTechnology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001, each of whichis hereby incorporated by reference. In certain instances, apeptidomimetic can be modified to create a constrained peptide thatadopts a distinct and specific preferred conformation, which canincrease the potency and selectivity of the peptide. For example, theconstrained peptide can be an azapeptide (Gante in Synthesis 1989,405-413). An azapeptide is synthesized by replacing the α-carbon of anamino acid with a nitrogen atom without changing the structure of theamino acid side chain. For example, the azapeptide can be synthesized byusing hydrazine in traditional peptide synthesis coupling methods, suchas by reacting hydrazine with a “carbonyl donor,” e.g.,phenylchloroformate.

Conjugation with Ligands to Promote Entry into Cells

Oligonucleotide agents can be modified to enhance entry into cells,e.g., an endocytic or non-endocytic mechanism. A ligand that increasescell permeability can be attached to an oligonucleotide agent in anumber of ways. One example of ligand attachment is by bonding to anSRMS, e.g., pyrroline-based SRMS.

In one embodiment, an oligonucleotide can be conjugated to apolyarginine that will enhance uptake into a wide range of cell-types.While not being bound by theory, the enhanced uptake is believed to beby a nonendocytic route. In another embodiment, an oligonucleotide canbe conjugated to a guanidium analog of an aminoglycoside to promote cellpermeability.

In another embodiment, an oligonucleotide can be conjugated with alipophilic moiety. The lipophilic moiety can be attached at the nitrogenatom of a pyrroline-based SRMS. Examples of lipophilic moieties includecholesterols, lipid, oleyl, retinyl, or cholesteryl residues. Otherlipophilic moieties include cholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine. Cholesterol is a particularly preferredexample.

The ligand that enhances cell permeability can be attached at the3′-terminus, the 5′-terminus, or internally. The ligand can be attachedto an SRMS, e.g., a pyrroline-based SRMS at the 3′-terminus, the5′-terminus, or at an internal linkage. The attachment can be direct orthrough a tethering molecule. Tethers, spacers, or linkers discussedherein can be used to attach the moiety to the SRMS.

Synthesis of Ligand-Conjugated Oligonucleotides of the Invention

The oligonucleotide compounds of the invention can be prepared usingsolution-phase or solid-phase organic synthesis. Organic synthesisoffers the advantage that the oligonucleotide strands comprisingnon-natural or modified nucleotides can be easily prepared. Importantly,pyrimidine nucleotides that have been modified at the C5-position can beincorporated into the oligonucleotide sequence. The double-strandedoligonucleotide compounds of the invention comprising non-naturalnucleobases and optionally non-natural sugar moieties may be preparedusing a two-step procedure. First, the individual strands of thedouble-stranded molecule are prepared separately. Then, the componentstrands are annealed.

The ligand-conjugated oligonucleotides of the invention may besynthesized by the use of an oligonucleotide that bears a pendantreactive functionality, such as that derived from the attachment of alinking molecule onto the oligonucleotide. This reactive oligonucleotidemay be reacted directly with commercially-available ligands, ligandsthat are synthesized bearing any of a variety of protecting groups, orligands that have a linking moiety attached thereto. The methods of thepresent invention facilitate the synthesis of ligand-conjugatedoligonucleotides by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the present invention via reaction of a selectedserum-binding ligand with a linking moiety located on the 5′ position ofa nucleoside or oligonucleotide. In certain instances, anoligonucleotide bearing a ligand attached to the 3′-terminus of theoligonucleotide is prepared by first covalently attaching a monomerbuilding block to a controlled-pore-glass support via a long-chainaminoalkyl group. Then, nucleotides are bonded via standard solid-phasesynthesis techniques to the monomer building-block bound to the solidsupport. The monomer building block may be a nucleoside or other organiccompound that is compatible with solid-phase synthesis.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents: U.S. Pat.Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawnto, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of thepresent invention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In one preferred embodiment of the methods of the invention, thepreparation of ligand-conjugated oligonucleotides commences with theselection of appropriate precursor molecules upon which to construct theligand molecule. Typically, the precursor is an appropriately-protectedderivative of the commonly-used nucleosides. For example, the syntheticprecursors for the synthesis of the ligand-conjugated oligonucleotidesof the present invention include, but are not limited to,2′-aminoalkoxy-5′-ODMT-nucleosides,2′-6-aminoalkylamino-5′-ODMT-nucleosides,5′-6-aminoalkoxy-2′-deoxy-nucleosides,5′-6-aminoalkoxy-2-protected-nucleosides,3′-6-aminoalkoxy-5′-ODMT-nucleosides, and3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in thenucleobase portion of the molecule. Methods for the synthesis of suchamino-linked protected nucleoside precursors are known to those ofordinary skill in the art.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

Representative hydroxylprotecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Furtherhydroxylprotecting groups, as well as other representative protectinggroups, are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y, 1991.

Examples of hydroxylprotecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the present invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

Therapeutic Uses for Compounds of the Invention

In a preferred embodiment of the present invention, the ligand attachedto the nucleoside enhances the pharmacokinetic properties of theoligonucleotide therapeutic or diagnostic agent. In a preferredembodiment, the oligonucleotide is a siRNA. Such improvedpharmacokinetic properties include increased binding of the antisensecompound to serum proteins, increased plasma concentration of theantisense compound, increased tissue distribution, increased capacity ofbinding of the antisense compound to serum proteins, and increasedhalf-lives.

The present invention provides a method for increasing the concentrationof an oligonucleotide in serum. According to such methods, the ligand isconjugated to an oligonucleotide, thus forming a conjugatedoligonucleotide. This conjugated oligonucleotide is then added to theserum.

The present invention further provides methods for increasing thecapacity of serum for an oligonucleotide. According to such methods, theligand is conjugated to an oligonucleotide, thus forming a conjugatedoligonucleotide. This conjugated oligonucleotide is then added to theserum.

The present invention also provides methods for increasing the bindingof an oligonucleotide to a portion of the vascular system. According tosuch methods, a vascular protein is selected which resides, in part, inthe circulating serum and, in part, in the non-circulating portion ofthe vascular system. Then, the ligand, e.g., naproxen, is conjugated toan oligonucleotide to form a conjugated oligonucleotide, which is thenadded to the vascular system.

The present invention further provides methods for promoting thecellular uptake of an oligonucleotide in a cell. According to suchmethods, a cellular protein is selected. This cellular protein is aprotein that resides on the cellular membrane and extends, in part,extracellularly so that part of this cellular protein extends onto theexternal side of the cellular membrane. Next, the ligand is conjugatedto an oligonucleotide to form a conjugated oligonucleotide. Thisconjugated oligonucleotide is then brought into contact with cells inwhich cellular uptake of the oligonucleotide is to be promoted. In apreferred embodiment, the oligonucleotide is an siRNA.

The present invention also provides methods of increasing cellularuptake of an oligonucleotide comprising contacting an organism with anoligonucleotide of the invention, said oligonucleotide being conjugatedto a ligand.

In one preferred embodiment of the invention, the protein targeted bythe oligonucleotide is a serum protein. It is preferred that the serumprotein targeted by the ligand-conjugated oligonucleotide compound is animmunoglobulin (an antibody). Preferred immunoglobulins areimmunoglobulin G and immunoglobulin M. Immunoglobulins are known toappear in blood serum and tissues of vertebrate animals.

In another embodiment of the invention, the serum protein targeted bythe oligonucleotide is a lipoprotein. Lipoproteins are blood proteinshaving molecular weights generally above 20,000 that carry lipids andare recognized by specific cell-surface receptors. The association withlipoproteins in the serum will initially increase pharmacokineticparameters such as half-life and distribution. A secondary considerationis the ability of lipoproteins to enhance cellular uptake viareceptor-mediated endocytosis.

In yet another embodiment, the serum protein targeted by theligand-conjugated oligonucleotide compound is α-2-macroglobulin. In yeta further embodiment, the serum protein targeted by a ligand-conjugatedoligonucleotide is α-1-glycoprotein.

Genes and Diseases

One aspect of the invention relates to a method of treating a subject atrisk for or afflicted with unwanted cell proliferation, e.g., malignantor nonmalignant cell proliferation. The method comprises providing aligand-conjugated oligonucleotide agent, wherein the oligonucleotide ishomologous to and can silence, e.g., by cleavage, a gene which promotesunwanted cell proliferation; and administering a therapeuticallyeffective dose of the ligand-conjugated oligonucleotide agent to asubject, preferably a human subject.

In a preferred embodiment the gene is a growth factor or growth factorreceptor gene, a kinase, e.g., a protein tyrosine, serine or threoninekinase gene, an adaptor protein gene, a gene encoding a G proteinsuperfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the oligonucleotide agent silences the PDGFbeta gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted PDGF beta expression, e.g.,testicular and lung cancers.

In another preferred embodiment the oligonucleotide agent silences theErb-B gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Erb-B expression, e.g., breastcancer.

In a preferred embodiment the oligonucleotide agent silences the Srcgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the oligonucleotide agent silences the CRKgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted CRK expression, e.g., colon and lungcancers.

In a preferred embodiment the oligonucleotide agent silences the GRB2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted GRB2 expression, e.g., squamous cellcarcinoma.

In another preferred embodiment the oligonucleotide agent silences theRAS gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted RAS expression, e.g., pancreatic,colon and lung cancers, and chronic leukemia.

In another preferred embodiment the oligonucleotide agent silences theMEKK gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted MEKK expression, e.g., squamouscell carcinoma, melanoma or leukemia.

In another preferred embodiment the oligonucleotide agent silences theJNK gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted JNK expression; e.g., pancreatic orbreast cancers.

In a preferred embodiment the oligonucleotide agent silences the RAFgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted RAF expression, e.g., lung cancer orleukemia.

In a preferred embodiment the oligonucleotide agent silences the Erk1/2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the oligonucleotide agent silences thePCNA(p21) gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted PCNA expression, e.g.,lung cancer.

In a preferred embodiment the oligonucleotide agent silences the MYBgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted MYB expression, e.g., colon cancer orchronic myelogenous leukemia.

In a preferred embodiment the oligonucleotide agent silences the c-MYCgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted c-MYC expression, e.g., Burkitt'slymphoma or neuroblastoma.

In another preferred embodiment the oligonucleotide agent silences theJUN gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted JUN expression, e.g., ovarian,prostate or breast cancers.

In another preferred embodiment the oligonucleotide agent silences theFOS gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted FOS expression, e.g., skin orprostate cancers.

In a preferred embodiment the oligonucleotide agent silences the BCL-2gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted BCL-2 expression, e.g., lung orprostate cancers or Non-Hodgkin lymphoma.

In a preferred embodiment the oligonucleotide agent silences the CyclinD gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin D expression, e.g., esophagealand colon cancers.

In a preferred embodiment the oligonucleotide agent silences the VEGFgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted VEGF expression, e.g., esophageal andcolon cancers.

In a preferred embodiment the oligonucleotide agent silences the EGFRgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the oligonucleotide agent silences theCyclin A gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Cyclin A expression, e.g., lungand cervical cancers.

In another preferred embodiment the oligonucleotide agent silences theCyclin E gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Cyclin E expression, e.g., lungand breast cancers.

In another preferred embodiment the oligonucleotide agent silences theWNT-1 gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted WNT-1 expression, e.g., basalcell carcinoma.

In another preferred embodiment the oligonucleotide agent silences thebeta-catenin gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted beta-catenin expression,e.g., adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the oligonucleotide agent silences thec-MET gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted c-MET expression, e.g.,hepatocellular carcinoma.

In another preferred embodiment the oligonucleotide agent silences thePKC gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted PKC expression, e.g., breastcancer.

In a preferred embodiment the oligonucleotide agent silences the NFKBgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the oligonucleotide agent silences the STAT3gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted STAT3 expression, e.g., prostatecancer.

In another preferred embodiment the oligonucleotide agent silences thesurvivin gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted survivin expression, e.g.,cervical or pancreatic cancers.

In another preferred embodiment the oligonucleotide agent silences theHer2/Neu gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Her2/Neu expression, e.g.,breast cancer.

In another preferred embodiment the oligonucleotide agent silences thetopoisomerase I gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted topoisomerase Iexpression, e.g., ovarian and colon cancers.

In a preferred embodiment the oligonucleotide agent silences thetopoisomerase II alpha gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted topoisomeraseII expression, e.g., breast and colon cancers.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p73 gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p73 expression, e.g.,colorectal adenocarcinoma.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p21(WAF1/CIP1) gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwantedp21(WAF1/CIP1) expression, e.g., liver cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p27(KIP1) gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p27(KIP1) expression,e.g., liver cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the PPM1D gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted PPM1D expression, e.g.,breast cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the RAS gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted RAS expression, e.g.,breast cancer.

In another preferred embodiment the oligonucleotide agent silencesmutations in the caveolin I gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwantedcaveolin I expression, e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the oligonucleotide agent silencesmutations in the MIB I gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted MIB Iexpression, e.g., male breast carcinoma (MBC).

In another preferred embodiment the oligonucleotide agent silencesmutations in the MTAI gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted MTAIexpression, e.g., ovarian carcinoma.

In another preferred embodiment the oligonucleotide agent silencesmutations in the M68 gene, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted M68expression, e.g., human adenocarcinomas of the esophagus, stomach,colon, and rectum.

In preferred embodiments the oligonucleotide agent silences mutations intumor suppressor genes, and thus can be used as a method to promoteapoptotic activity in combination with chemotherapeutics.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p53 tumor suppressor-gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted p53expression, e.g., gall bladder, pancreatic and lung cancers.

In a preferred embodiment the oligonucleotide agent silences mutationsin the p53 family member DN-p63, and thus can be used to treat a subjecthaving or at risk for a disorder characterized by unwanted DN-p63expression, e.g., squamous cell carcinoma.

In a preferred embodiment the oligonucleotide agent silences mutationsin the pRb tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted pRbexpression, e.g., oral squamous cell carcinoma.

In a preferred embodiment the oligonucleotide agent silences mutationsin the APC1 tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted APC1expression, e.g., colon cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the BRCA1 tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted BRCA1expression, e.g., breast cancer.

In a preferred embodiment the oligonucleotide agent silences mutationsin the PTEN tumor suppressor gene, and thus can be used to treat asubject having or at risk for a disorder characterized by unwanted PTENexpression, e.g., hamartomas, gliomas, and prostate and endometrialcancers.

In a preferred embodiment the oligonucleotide agent silences MLL fusiongenes, e.g., MLL-AF9, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted MLL fusion geneexpression, e.g., acute leukemias.

In another preferred embodiment the oligonucleotide agent silences theBCR/ABL fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted BCR/ABL fusion geneexpression, e.g., acute and chronic leukemias.

In another preferred embodiment the oligonucleotide agent silences theTEL/AML1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted TEL/AML1 fusion geneexpression, e.g., childhood acute leukemia.

In another preferred embodiment the oligonucleotide agent silences theEWS/FLI1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted EWS/FLI1 fusion geneexpression, e.g., Ewing Sarcoma.

In another preferred embodiment the oligonucleotide agent silences theTLS/FUS1 fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted TLS/FUS1 fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the oligonucleotide agent silences thePAX3/FKHR fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted PAX3/FKHR fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the oligonucleotide agent silences theAML1/ETO fusion gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted AML1/ETO fusion geneexpression, e.g., acute leukemia.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder that may benefit by angiogenesis inhibition e.g., cancer. Themethod comprises providing a ligand-conjugated oligonucleotide agent,wherein said oligonucleotide agent is homologous to and can silence,e.g., by cleavage, a gene which mediates angiogenesis; and administeringa therapeutically effective dosage of said ligand-conjugatedoligonucleotide agent to a subject, preferrably a human.

In a preferred embodiment the oligonucleotide agent silences the alphav-integrin gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted alpha V integrin, e.g.,brain tumors or tumors of epithelial origin.

In a preferred embodiment the oligonucleotide agent silences the Flt-1receptor gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted Flt-1 receptors, eg. Cancer andrheumatoid arthritis.

In a preferred embodiment the oligonucleotide agent silences the tubulingene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted tubulin, eg. Cancer and retinalneovascularization.

Another aspect of the invention relates to a method of treating asubject infected with a virus or at risk for or afflicted with adisorder or disease associated with a viral infection. The methodcomprises providing a ligand-conjugated oligonucleotide agent, whereinsaid oligonucleotide agent is homologous to and can silence, e.g., bycleavage, a viral gene of a cellular gene which mediates viral function,e.g., entry or growth; and administering a therapeutically effectivedose of said ligand-conjugated oligonucleotide agent to a subject,preferably a human subject.

Thus, the invention provides for a method of treating patients infectedby the Human Papilloma Virus (HPV) or at risk for or afflicted with adisorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% ofcervical carcinomas and thus an antiviral therapy is an attractivemethod to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. Inanother preferred embodiment, the HPV gene is one of the group of E2,E6, or E7.

In a preferred embodiment the expression of a human gene that isrequired for HPV replication is reduced.

The invention also includes a method of treating patients infected bythe Human Immunodeficiency Virus (HIV) or at risk for or afflicted witha disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome(AIDS). In a preferred embodiment, the expression of a HIV gene isreduced. In another preferred embodiment, the HIV gene is CCR5, Gag, orRev. In a preferred embodiment the expression of a human gene that isrequired for HIV replication is reduced. In another preferredembodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected bythe Hepatitis B Virus (HBV) or at risk for or afflicted with a disordermediated by HBV, e.g., cirrhosis and heptocellular carcinoma. In apreferred embodiment, the expression of a HBV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of the tail region of the HBV core protein, the pre-cregious(pre-c) region, or the cregious (c) region. In another preferredembodiment, a targeted HBV-RNA sequence is comprised of the poly(A)tail.

In preferred embodiment the expression of a human gene that is requiredfor HBV replication is reduced.

The invention also provides for a method of treating patients infectedby the Hepatitis A Virus (HAV), or at risk for or afflicted with adisorder mediated by HAV. In a preferred embodiment the expression of ahuman gene that is required for HAV replication is reduced.

The present invention provides for a method of treating patientsinfected by the Hepatitis C Virus (HCV), or at risk for or afflictedwith a disorder mediated by HCV, e.g., cirrhosis. In a preferredembodiment, the expression of a HCV gene is reduced. In anotherpreferred embodiment the expression of a human gene that is required forHCV replication is reduced.

The present invention also provides for a method of treating patientsinfected by the any of the group of Hepatitis Viral strains comprisinghepatitis D, E, F, G, or H, or patients at risk for or afflicted with adisorder mediated by any of these strains of hepatitis. In a preferredembodiment, the expression of a Hepatitis, D, E, F, G, or H gene isreduced. In another preferred embodiment the expression of a human genethat is required for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected bythe Respiratory Syncytial Virus (RSV) or at risk for or afflicted with adisorder mediated by RSV, e.g, lower respiratory tract infection ininfants and childhood asthma, pneumonia and other complications, e.g.,in the elderly. In a preferred embodiment, the expression of a RSV geneis reduced. In another preferred embodiment, the targeted HBV geneencodes one of the group of genes N, L, or P. In a preferred embodimentthe expression of a human gene that is required for RSV replication isreduced.

Methods of the invention provide for treating patients infected by theHerpes Simplex Virus (HSV) or at risk for or afflicted with a disordermediated by HSV, e.g, genital herpes and cold sores as well aslife-threatening or sight-impairing disease mainly in immunocompromisedpatients. In a preferred embodiment, the expression of a HSV gene isreduced. In another preferred embodiment, the targeted HSV gene encodesDNA polymerase or the helicase-primase. In a preferred embodiment theexpression of a human gene that is required for HSV replication isreduced.

The invention also provides a method for treating patients infected bythe herpes Cytomegalovirus (CMV) or at risk for or afflicted with adisorder mediated by CMV, e.g., congenital virus infections andmorbidity in immunocompromised patients. In a preferred embodiment, theexpression of a CMV gene is reduced. In a preferred embodiment theexpression of a human gene that is required for CMV replication isreduced.

Methods of the invention also provide for a method of treating patientsinfected by the herpes Epstein Barr Virus (EBV) or at risk for orafflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma,non-Hodgkin lymphoma, and Hodgkin disease. In a preferred embodiment,the expression of a EBV gene is reduced. In a preferred embodiment theexpression of a human gene that is required for EBV replication isreduced.

Methods of the invention also provide for treating patients infected byKaposi's Sarcoma-associated Herpes Virus (KSHV), also called humanherpesvirus 8, or patients at risk for or afflicted with a disordermediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman'sdisease and AIDS-associated primary effusion lymphoma. In a preferredembodiment, the expression of a KSHV gene is reduced. In a preferredembodiment the expression of a human gene that is required for KSHVreplication is reduced.

The invention also includes a method for treating patients infected bythe JC Virus (JCV) or a disease or disorder associated with this virus,e.g., progressive multifocal leukoencephalopathy (PML). In a preferredembodiment, the expression of a JCV gene is reduced. In preferredembodiment the expression of a human gene that is required for JCVreplication is reduced.

Methods of the invention also provide for treating patients infected bythe myxovirus or at risk for or afflicted with a disorder mediated bymyxovirus, e.g., influenza. In a preferred embodiment, the expression ofa myxovirus gene is reduced. In a preferred embodiment the expression ofa human gene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe rhinovirus or at risk for of afflicted with a disorder mediated byrhinovirus, e.g., the common cold. In a preferred embodiment, theexpression of a rhinovirus gene is reduced. In preferred embodiment theexpression of a human gene that is required for rhinovirus replicationis reduced.

Methods of the invention also provide for treating patients infected bythe coronavirus or at risk for of afflicted with a disorder mediated bycoronavirus, e.g., the common cold. In a preferred embodiment, theexpression of a coronavirus gene is reduced. In preferred embodiment theexpression of a human gene that is required for coronavirus replicationis reduced.

Methods of the invention also provide for treating patients infected bythe flavivirus West Nile or at risk for or afflicted with a disordermediated by West Nile Virus. In a preferred embodiment, the expressionof a West Nile Virus gene is reduced. In another preferred embodiment,the West Nile Virus gene is one of the group comprising E, NS3, or NS5.In a preferred embodiment the expression of a human gene that isrequired for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe St. Louis Encephalitis flavivirus, or at risk for or afflicted witha disease or disorder associated with this virus, e.g., viralhaemorrhagic fever or neurological disease. In a preferred embodiment,the expression of a St. Louis Encephalitis gene is reduced. In apreferred embodiment the expression of a human gene that is required forSt. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Tick-borne encephalitis flavivirus, or at risk for or afflicted witha disorder mediated by Tick-borne encephalitis virus, e.g., viralhaemorrhagic fever and neurological disease. In a preferred embodiment,the expression of a Tick-borne encephalitis virus gene is reduced. In apreferred embodiment the expression of a human gene that is required forTick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patientsinfected by the Murray Valley encephalitis flavivirus, which commonlyresults in viral haemorrhagic fever and neurological disease. In apreferred embodiment, the expression of a Murray Valley encephalitisvirus gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for Murray Valley encephalitis virusreplication is reduced.

The invention also includes methods for treating patients infected bythe dengue flavivirus, or a disease or disorder associated with thisvirus, e.g., dengue haemorrhagic fever. In a preferred embodiment, theexpression of a dengue virus gene is reduced. In a preferred embodimentthe expression of a human gene that is required for dengue virusreplication is reduced.

Methods of the invention also provide for treating patients infected bythe Simian Virus 40 (SV40) or at risk for or afflicted with a disordermediated by SV40, e.g., tumorigenesis. In a preferred embodiment, theexpression of a SV40 gene is reduced. In a preferred embodiment theexpression of a human gene that is required for SV40 replication isreduced.

The invention also includes methods for treating patients infected bythe Human T Cell Lymphotropic Virus (HTLV), or a disease or disorderassociated with this virus, e.g., leukemia and myelopathy. In apreferred embodiment, the expression of a HTLV gene is reduced. Inanother preferred embodiment the HTLV1 gene is the Tax transcriptionalactivator. In a preferred embodiment the expression of a human gene thatis required for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflictedwith a disorder mediated by Mo-MuLV, e.g., T-cell leukemia. In apreferred embodiment, the expression of a Mo-MuLV gene is reduced. In apreferred embodiment the expression of a human gene that is required forMo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe encephalomyocarditis virus (EMCV) or at risk for or afflicted with adisorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditisin mice and pigs and is capable of infecting human myocardial cells.This virus is therefore a concern for patients undergoingxenotransplantation. In a preferred embodiment, the expression of a EMCVgene is reduced. In a preferred embodiment the expression of a humangene that is required for EMCV replication is reduced.

The invention also includes a method for treating patients infected bythe measles virus (MV) or at risk for or afflicted with a disordermediated by MV, e.g., measles. In a preferred embodiment, the expressionof a MV gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for MV replication is reduced.

The invention also includes a method for treating patients infected bythe Vericella zoster virus (VZV) or at risk for or afflicted with adisorder mediated by VZV, e.g. chicken pox or shingles (also calledzoster). In a preferred embodiment, the expression of a VZV gene isreduced. In a preferred embodiment the expression of a human gene thatis required for VZV replication is reduced.

The invention also includes a method for treating patients infected byan adenovirus or at risk for or afflicted with a disorder mediated by anadenovirus, e.g. respiratory tract infection. In a preferred embodiment,the expression of an adenovirus gene is reduced. In a preferredembodiment the expression of a human gene that is required foradenovirus replication is reduced.

The invention includes a method for treating patients infected by ayellow fever virus (YFV) or at risk for or afflicted with a disordermediated by a YFV, e.g. respiratory tract infection. In a preferredembodiment, the expression of a YFV gene is reduced. In anotherpreferred embodiment, the preferred gene is one of a group that includesthe E, NS2A, or NS3 genes. In a preferred embodiment the expression of ahuman gene that is required for YFV replication is reduced.

Methods of the invention also provide for treating patients infected bythe poliovirus or at risk for or afflicted with a disorder mediated bypoliovirus, e.g., polio. In a preferred embodiment, the expression of apoliovirus gene is reduced. In a preferred embodiment the expression ofa human gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected bya poxvirus or at risk for or afflicted with a disorder mediated by apoxvirus, e.g., smallpox. In a preferred embodiment, the expression of apoxvirus gene is reduced. In a preferred embodiment the expression of ahuman gene that is required for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subjectinfected with a pathogen, e.g., a bacterial, amoebic, parasitic, orfungal pathogen. The method comprises providing a ligand-conjugatedoligonucleotide agent, wherein said oligonucleotide is homologous to andcan silence, e.g., by cleavage of a pathogen gene; and administering atherapeutically effective dose of said ligand-conjugated oligonucleotideagent to a subject, prefereably a human subject.

The target gene can be one involved in growth, cell wall synthesis,protein synthesis, transcription, energy metabolism, e.g., the Krebscycle, or toxin production. Thus, the present invention provides for amethod of treating patients infected by a plasmodium that causesmalaria. In a preferred embodiment, the expression of a plasmodium geneis reduced. In another preferred embodiment, the gene is apical membraneantigen 1 (AMA1). In a preferred embodiment the expression of a humangene that is required for plasmodium replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium ulcerans, or a disease or disorder associated withthis pathogen, e.g. Buruli ulcers. In a preferred embodiment, theexpression of a Mycobacterium ulcerans gene is reduced. In a preferredembodiment the expression of a human gene that is required forMycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium tuberculosis, or a disease or disorder associated withthis pathogen, e.g. tuberculosis. In a preferred embodiment, theexpression of a Mycobacterium tuberculosis gene is reduced. In apreferred embodiment the expression of a human gene that is required forMycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium leprae, or a disease or disorder associated with thispathogen, e.g. leprosy. In a preferred embodiment, the expression of aMycobacterium leprae gene is reduced. In a preferred embodiment theexpression of a human gene that is required for Mycobacterium lepraereplication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Staphylococcus aureus, or a disease or disorder associatedwith this pathogen, e.g. infections of the skin and muscous membranes.In a preferred embodiment, the expression of a Staphylococcus aureusgene is reduced. In a preferred embodiment the expression of a humangene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pneumoniae, or a disease or disorderassociated with this pathogen, e.g. pneumonia or childhood lowerrespiratory tract infection. In a preferred embodiment, the expressionof a Streptococcus pneumoniae gene is reduced. In a preferred embodimentthe expression of a human gene that is required for Streptococcuspneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pyogenes, or a disease or disorder associatedwith this pathogen, e.g. Strep throat or Scarlet fever. In a preferredembodiment, the expression of a Streptococcus pyogenes gene is reduced.In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Chlamydia pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection. In a preferred embodiment, the expression of a Chlamydiapneumoniae gene is reduced. In a preferred embodiment the expression ofa human gene that is required for Chlamydia pneumoniae replication isreduced.

The invention also includes methods for treating patients infected bythe bacteria Mycoplasma pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection. In a preferred embodiment, the expression of a Mycoplasmapneumoniae gene is reduced. In a preferred embodiment the expression ofa human gene that is required for Mycoplasma pneumoniae replication isreduced.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder characterized by an unwanted immune response, e.g., aninflammatory disease or disorder, or an autoimmune disease or disorder.The method comprises providing a ligand-conjugated oligonucleotideagent, wherein said oligonucleotide agent is homologous to and cansilence, e.g., by cleavage, a gene which mediates an unwanted immuneresponse; and administering said ligand-conjugated oligonucleotide agentto a subject, preferrably a human subject. In a preferred embodiment thedisease or disorder is an ischemia or reperfusion injury, e.g., ischemiaor reperfusion injury associated with acute myocardial infarction,unstable angina, cardiopulmonary bypass, surgical intervention e.g.,angioplasty, e.g., percutaneous transluminal coronary angioplasty, theresponse to a transplantated organ or tissue, e.g., transplanted cardiacor vascular tissue; or thrombolysis. In a preferred embodiment thedisease or disorder is restenosis, e.g., restenosis associated withsurgical intervention e.g., angioplasty, e.g., percutaneous transluminalcoronary angioplasty. In a prefered embodiment the disease or disorderis Inflammatory Bowel Disease, e.g., Crohn Disease or UlcerativeColitis. In a prefered embodiment the disease or disorder isinflammation associated with an infection or injury. In a preferedembodiment the disease or disorder is asthma, lupus, multiple sclerosis,diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid orpsoriatic. In particularly preferred embodiments the oligonucleotideagent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.In particularly preferred embodiments the oligonucleotide agent silencesa selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM),I-selectin, P-selectin glycoprotein-1 (PSGL-1). In particularlypreferred embodiments the oligonucleotide agent silences a component ofthe complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5convertase.

In particularly preferred embodiments the oligonucleotide agent silencesa chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1 I, IL-1J, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the oligonucleotide agent silences GCSF, Gro1,Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J,RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1,I-309.

Another aspect of the invention features, a method of treating asubject, e.g., a human, at risk for or afflicted with acute pain orchronic pain. The method comprises providing a ligand-conjugatedoligonucleotide agent, wherein said ligand is an aromatic group and saidoligonucleotide is homologous to and can silence, e.g., by cleavage, agene which mediates the processing of pain; and administering atherapeutically effective dose of said ligand-conjugated oligonucleotideagent to a subject, preferrably a human subject. In particularlypreferred embodiments the oligonucleotide agent silences a component ofan ion channel. In particularly preferred embodiments theoligonucleotide agent silences a neurotransmitter receptor or ligand.

Another aspect of the invention relates to a method of treating asubject, e.g., a human, at risk for or afflicted with a neurologicaldisease or disorder. The method comprises providing a ligand-conjugatedoligonucleotide agent, wherein said ligand is an aromtic group and saidoligonucleotide is homologous to and can silence, e.g., by cleavage, agene which mediates a neurological disease or disorder; andadministering a therapeutically effective dose of said ligand-conjugatedoligonucleotide agent the to a subject, preferrably a human. In aprefered embodiment the disease or disorder is Alzheimer Disease orParkinson Disease. In particularly preferred embodiments theoligonucleotide agent silences an amyloid-family gene, e.g., APP; apresenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein. In a preferredembodiment the disease or disorder is a neurodegenerative trinucleotiderepeat disorder, e.g., Huntington disease, dentatorubral pallidoluysianatrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3(Machado-Joseph disease), SCA7 or SCA8.

In particularly preferred embodiments the oligonucleotide agent silencesHD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in euploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific cleavage orsilencing of one allele of an essential gene with a ligand-conjugatedoligonucleotide agent of the invention. The oligonucleotide agent isselected such that it targets the single allele of the essential genefound in the cells having LOH but does not silence the other allele,which is present in cells which do not show LOH. In essence, itdiscriminates between the two alleles, preferentially silencing theselected allele. In essence polymorphisms, e.g., SNPs of essential genesthat are affected by LOH, are used as a target for a disordercharacterized by cells having LOH, e.g., cancer cells having LOH. E.g.,one of ordinary skill in the art can identify essential genes which arein proximity to tumor suppressor genes, and which are within a LOHregion which includes the tumor suppressor gene. The gene encoding thelarge subunit of human RNA polymerase II, POLR2A, a gene located inclose proximity to the tumor suppressor gene p53, is such a gene. Itfrequently occurs within a region of LOH in cancer cells. Other genesthat occur within LOH regions and are lost in many cancer cell typesinclude the group comprising replication protein A 70-kDa subunit,replication protein A 32-kD, ribonucleotide reductase, thymidilatesynthase, TATA associated factor 2H, ribosomal protein S14, eukaryoticinitiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase,NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, another aspect of the invention relates to a method oftreating a disorder characterized by LOH, e.g., cancer. The methodcomprises optionally, determining the genotype of the allele of a genein the region of LOH and preferably determining the genotype of bothalleles of the gene in a normal cell; providing a ligand-conjugatedoligonucleotide agent which preferentially cleaves or silences theallele found in the LOH cells; and administering a therapeuticallyeffective dose of said ligand-conjugated oligonucleotide agent to thesubject, preferrably a human.

The invention also includes a ligand-conjugated oligonucleotide agentdisclosed herein, e.g, an oligonucleotide agent which can preferentiallysilence, e.g., cleave, one allele of a polymorphic gene.

In another aspect, the invention provides a method of cleaving orsilencing more than one gene with a ligand-conjugated oligonucleotideagent. In these embodiments the oligonucleotide agent is selected sothat it has sufficient homology to a sequence found in more than onegene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO: 1)isconserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 andlamin A/C. Thus an oligonucleotide agent targeted to this sequence wouldeffectively silence the entire collection of genes.

The invention also includes a ligand-conjugated oligonucleotide agent,which can silence more than one gene.

In a preferred embodiment, the oligonucleotide is a siRNA.

Compounds of the Invention

The compounds of the invention relate to ligand-bearingoligonucleotides, wherein the ligand is covalently attached to theC5-position of a pyrimidine-bearing nucleotide. In certain embodiments,the compounds of the invention relate to a double-strandedoligonucleotide sequence, wherein a ligand is bound to at least onenucleoside in only one of the two strands. In certain embodiments, thecompounds of the invention relate to a double-stranded oligonucleotidesequence, wherein at least one nucleoside in both of the strandscomprises a bound ligand. In a preferred embodiment, the oligonucleotideis a siRNA. In certain embodiments, the present invention relates to asingle-stranded oligonucleotide comprising at least one ligand. Theligand improves the pharmacokinetic properties of the oligonucleotide.

The nucleosides are linked by phosphorus-containing ornon-phosphorus-containing covalent internucleoside linkages. For thepurposes of identification, ligand-conjugated nucleosides can becharacterized as ligand-bearing nucleosides or ligand-nucleosideconjugates. The linked nucleosides having ligands conjugated to anucleoside within their sequence will demonstrate enhanced biologicalactivity when compared to like oligonucleotides that are not conjugated.

The ligand-conjugated oligonucleotides of the present invention alsoinclude conjugates of oligonucleotides and linked nucleosides whereinthe ligand is attached directly to the nucleoside or nucleotide whereinthe linker group is just a bond. The ligand may preferably be attached,via linking groups, at a carboxyl, amino or oxo group of the ligand.Typical linking groups may be ester, amide or carbamate groups.

Specific examples of preferred modified nucleotides envisioned for usein the ligand-conjugated oligonucleotides of the present inventioninclude nucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined here, oligonucleotides havingmodified backbones or internucleoside linkages include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes the invention,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified, and in fact more than one of the following modifications maybe incorporated in a single siRNA compound or even in a singlenucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalklyphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States Patents that teach the preparation of theabove phosphorus atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

Some preferred embodiments of the present invention employoligonucleotides with phosphorothioate linkages and oligonucleosideswith heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsopreferred are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

The oligonucleotides employed in the ligand-conjugated oligonucleotidesof the present invention may additionally or alternatively comprisenucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

The oligonucleotides employed in the ligand-conjugated oligonucleotidesof the present invention may additionally or alternatively comprise oneor more substituted sugar moieties. Preferred oligonucleotides compriseone of the following at the 2′ position: OH; F; O—, S—, or N-alkyl, O—,S—, or N-alkenyl, or O, S— or N-alkynyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. a further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook (Anti-CancerDrug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” herebyincorporated by reference in its entirety.

Additional sugar substituent groups amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, wherein each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No.5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in itsentirety. The incorporation of 2′-SR monomer synthons is disclosed byHamm et al. (J. Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides aredisclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; andPolushin et al., Tetrahedron Lett., 1996, 37, 3227-3230. Furtherrepresentative 2′-substituent groups amenable to the present inventioninclude those having one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃ is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, filed Jul. 27, 1998, entitled “RNATargeted 2′-Modified Oligonucleotides that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring 0 include,but are not limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarsstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,700,920; and 5,859,221, all of which are hereby incorporated byreference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one additional modification of theligand-conjugated oligonucleotides of the present invention involveschemically linking to the oligonucleotide one or more additionalnon-ligand moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties, such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

The present invention also includes compositions employingoligonucleotides that are substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

The present invention further encompasses ligand-conjugatedoligonucleotides employing ribozymes. Synthetic RNA molecules andderivatives thereof that catalyze highly specific endoribonucleaseactivities are known as ribozymes. (See, generally, U.S. Pat. No.5,543,508 to Haseloff et al., issued Aug. 6, 1996, and U.S. Pat. No.5,545,729 to Goodchild et al., issued Aug. 13, 1996.) The cleavagereactions are catalyzed by the RNA molecules themselves. In naturallyoccurring RNA molecules, the sites of self-catalyzed cleavage arelocated within highly conserved regions of RNA secondary structure(Buzayan et al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 8859; Forsteret al., Cell, 1987, 50, 9). Naturally occurring autocatalytic RNAmolecules have been modified to generate ribozymes which can be targetedto a particular cellular or pathogenic RNA molecule with a high degreeof specificity. Thus, ribozymes serve the same general purpose asantisense oligonucleotides (i.e., modulation of expression of a specificgene) and, like oligonucleotides, are nucleic acids possessingsignificant portions of single-strandedness. That is, ribozymes havesubstantial chemical and functional identity with oligonucleotides andare thus considered to be equivalents for purposes of the presentinvention.

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:111; Kabanov et al.,FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such oligonucleotideconjugates have been listed above. Typical conjugation protocols involvethe synthesis of oligonucleotides bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with theoligonucleotide still bound to the solid support or following cleavageof the oligonucleotide in solution phase. Purification of theoligonucleotide conjugate by HPLC typically affords the pure conjugate.

Alternatively, the molecule being conjugated may be converted into abuilding block, such as a phosphoramidite, via an alcohol group presentin the molecule or by attachment of a linker bearing an alcohol groupthat may be phosphitylated.

Importantly, each of these approaches may be used for the synthesis ofligand-conjugated oligonucleotides. Aminolinked oligonucleotides may becoupled directly with ligand via the use of coupling reagents orfollowing activation of the ligand as an NHS or pentfluorophenolateester. Ligand phosphoramidites may be synthesized via the attachment ofan aminohexanol linker to one of the carboxyl groups followed byphosphitylation of the terminal alcohol functionality. Other linkers,such as cysteamine, may also be utilized for conjugation to achloroacetyl linker present on a synthesized oligonucleotide.

For the purposes of illustration, the ligand-bearing nucleotide can bedivided into three regions: ligand, linker, and nucleotide. In thepresent invention, the ligand is bound to the nucleotide via a linker.The purpose of the linker is to covalently attach the ligand to thenucleotide. The structure of the linker is dictated by the functionalgroup used to bind the ligand. In a preferred embodiment, the linker isamenable to solid phase synthesis techniques. A more detailed discussionof each of the variable regions is presented below.

Ligand

In the present invention, the ligand can be a wide variety of organiccompounds which impart improved pharmacological properties to theoligonucleotde when the ligand(s) is attached to the oligonucleotide.For example, the ligand can be a steroid, bile acid, lipid, folic acid,pyridoxal, B12, riboflavin, biotin, aromatic compound, polycycliccompound, crown ether, intercalator, cleaver molecule, protein-bindingagent, or carbohydrate. In certain embodiments, the steroid ischolesterol. The ligand can also be a vitamin, peptide, terpene, oramino acid.

A large number of steroids are known in the art and are amenable to thepresent invention. Representative examples of steriods includecholesterol, 5β-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, and budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

A large number of bile acids are known in the art and are amenable tothe present invention. Bile acids occur in conjugation with glycine ortaurine in bile of most vertebrates and some of them find use inmedicine. Thus, some bile acids—due to their inherent pharmacologicalproperties—are used as cholerectics (see, for example, James E. F.Reynolds (editor) Martindale The Extra Pharmacopoeia, 30^(th) Edition,The Pharmaceutical Press, London (1993), page 1341). Representativeexamples of bile acids include cholic acid, deoxycholic acid,taurocholic acid, glycocholic acid, glycodeoxycholic acid,taurodeoxycholic acid, ursodeoxycholic acid, and chenodeoxycholic acid.Additional bile acids amenable to the present invention include thosedescribed in U.S. Pat. Nos. 5,641,767; 5,656,277; 5,610,151; 5,428,182;and 3,910,888.

A large number of lipids are known in the art and are amenable to thepresent invention. Representative examples of lipids include lauricacid, myristic acid, palmitic acid, stearic acid, arachidic acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, triacylglycerols, phosphoacylglycerols, sphingolipids,monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes,and tetraterpenes.

A large number of aromatic compounds are known in the art and areamenable to the present invention. Representative examples of aromaticcompounds include optionally substituted phenyl, naphthyl, anthracenyl,phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl,phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl,quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl,benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl,pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl,tetrazolyl, and furanyl.

A large number of carbohydrates are known in the art and are amenable tothe present invention. Representative examples of carbohydrates includeerythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose,glucose, mannose, gulose, idose, galactose, and talose; or adisaccharide or trisaccharide formed via a 1,4 glycoside linkage betweenany of them. In certain instances, the carbohydrate is a hexose orpentose. In certain instances, the carbohydrate is selected from one ofthe carbohydrates shown below.

In certain instances, the carbohydrates mentioned above have ancarboxyalkyl tether attached to the glycosyl oxygen atom. For proceduresrelating to the preparation of carbohydrates, such as those describedabove, bearing a carboxyalkyl attached to the glycosyl oxygen atom seeValentijn et al. Tetrahedron 1997, 53, 759; Wijsman et al. Recueil desTravaux Chimiques des Pays-Bas, 1996, 115, 397; and scheme 1.

A large number of polycyclic compounds are known in the art and areamenable to the present invention. Representative classes of polycycliccompounds include bicyclic compounds wherein, the first and second ringare independently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, both rings are saturated. In certaininstances, the first ring contains no heteroatoms. In certain instances,the second ring contains to heteroatoms. In certain instances, the firstring contains a nitrogen atom. In certain instances, the second ringcontains a nitrogen atom. In certain instances, the polycyclic compoundis a tricyclic compound, wherein the first, second, and third ring areindependently a 3, 4, 5, or 6-member saturated or unsaturated carbonring containing 0, 1, 2, or 3 hetereoatoms selected from the groupconsisting of O, N, or S. In certain instances, the first ring is anaromatic ring. In certain instances, the second ring is an aromaticring. In certain instances, the third ring is an aromatic ring. Incertain instances, all three rings are saturated. In certain instances,the first ring contains no heteroatoms. In certain instances, the secondring contains to heteroatoms. In certain instances, the third ringcontains to heteroatoms. In certain instances, the first ring contains anitrogen atom. In certain instances, the second ring contains a nitrogenatom. In certain instances, the third ring contains a nitrogen atom. Incertain instances, the polycyclic compound is a bridged polycycliccompound. In certain instances, the polycyclic compound is abicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.1]octane,bicyclo[3.2.2]nonane, or bicyclo[3.3.1]nonane.

A large number of crown ethers are known in the art and are amenable tothe present invention. Crown ethers are macrocyclic, polyether, neutralcompounds containing 4-20 oxygen atoms each separated from the next bytwo or more carbon atoms. Macrocyclic polyethers have been found to formstable complexes with salts of alkali metals and other metals andammonium salts; “Macrocyclic polyethers and their complexes”, C. J.Pederson et al, Angew. Chem. Intern. Ed., Vol. 11, page 16, (1972) andU.S. Pat. Nos. 3,562,295 and 3,687,978. Since the stereo models ofmacrocyclic polyethers give a crown-like appearance, they are commonlydesignated as N-crown-M polyethers, wherein N is the total number ofatoms in the polyether ring and M is the number of oxygen atoms in thepolyether ring. Crown polyethers ranging in size from cyclic tetramersof ethylene oxide ([12]-crown-4) and propylene oxide ([16]-crown-4) to60-membered polyether rings (dibenzo [60]-crown-20) have been reported.Preferred crown ethers include 12-crown-4,15-crown-5, and 18-crown-6.

A large number of oligonucleotide intercalators are known in the art andare amenable to the present invention. One class of intercalators areDNA intercalators which bind noncovalently to duplex DNA and arecharacterized by a flat molecule which inserts between base pairs of thedouble helix of DNA. Representative examples of intercalators includep-carboxy methidium, p-carboxy ethidium, acridine and ellipticine.

A large number of oligonucleotide cleaver molecules are known in the artand are amenable to the present invention. A cleaver molecule is acompound that can sever an oligonucleotide strand. Bleomycin, aglycopeptide antibiotic, is known to bind to and cleave DNA in areaction that depends on the presence of ferrous ion and molecularoxygen, “Bleomycin: Chemical, Biochemical and Biological Aspects”;Hecht, S. M., Ed.; Springer Verlag: New York, 1979; Sausville, E. A.;Peisach, J.; Horwitz, S. B. “Biochemistry” 1978, 17, 2740. Burger, R.M.; Peisach, J; Horwitz, S. B. “Life Sciences” 1981, 28, 715; and Lown,J. W.; Sim, S. F. “Biochem. Biophys. Res. Comm.” 1977, 77, 1150. Theantitumor agent streptonigrin is also capable of causing single strandbreaks in DNA using oxygen and cuprous ion, Cone, R; Hasan, S. K.; Lown,J. W.; Morgan, A. R. “Can. J. Biochem.” 1976, 54, 219. Recently, the1-10 phenanthroline-cuprous complex has been shown to cleave DNA in thepresence of oxygen, Sigman, D. S.; Graham, D. R.; D'Aurora, V.; Stern,A. M. “J. Biol. Chem.” 1979, 254, 12269; Graham, D. R.; Marshall, L. E.;Reich, K. A.; Sigman, D. S. “J. Amer. Chem. Soc.” 1980, 102, 5419;Marshall, L. E.; Graham, D. R.; Reich, K. A.; Sigman, D. S.“Biochemistry” 1981, 20, 244; and Que, B. G.; Downey, K. M.; So., A. G.“Biochemistry” 1980, 19, 5987. In addition, methidium, ethidium, andcisplatin are known to cleave oligonucleotide sequences.

A large number of saturated 5-membered rings are known in the art andare amenable to the present invention. Preferred saturated 5-memberedrings are optionally substituted cyclopentane, pyrrolidine,tetrahydrofuran, tetrahydrothiophene, and 1,1-difluorocyclopentane.

In certain instances, the ligand is selected from one of the radicalsshown below.

Linker

In a preferred embodiment of the invention, the ligand is attached to anoligonucleotide via a linking group, to form a ligand-conjugatedoligonucleotide. Preferred linkers of the invention include, but are notlimited to, alkyl linkers, alkenyl linkers, α,β-unsaturated amidelinkers, α,β-unsaturated ester linkers, ketone linkers, thioetherlinkers, aminoalkyl linkers, 6-aminoalkoxy linkers, 6-aminoalkylaminolinkers, cysteamine, heterobifunctional linkers, homobifunctionallinkers, and a universal tether (derived from3-dimethoxytrityloxy-2-aminopropanol). A particularly preferred tetherfor the synthesis of ligand conjugated oligonucleotides of the inventionis an α,β-unsaturated amide linker. A variety of heterobifunctional andhomobifunctional tethers are available from Pierce Co. (Rockford, Ill.).Such heterobifunctional and homobifunctional tethers are particularlyuseful in conjunction with the 6-aminoalkoxy and 6-aminoalkylaminomoieties to form extended tethers useful for linking ligands to anucleoside.

In certain instances, conjugation of ligand molecules is achieved byconjugation of the ligand to an amino tether on the nucleoside. This canbe effected in several ways. For example, a ligand-nucleoside conjugateof the invention can be prepared by conjugation of the ligand moleculeto the nucleoside using EDC/sulfo-NHS (i.e.1-ethyl-3(3-dimethylaminopropylcarbodiimide/N-hydroxysulfosuccinimide)to conjugate the carboxylate function of the ligand with the aminofunction of the linking group on the nucleoside.

The ligand-conjugated oligonucleotides of the present invention may beprepared by conjugation of the ligand molecule to the nucleosidesequence via a heterobifunctional tether such asm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS) or succinimidyl4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate (SMCC), to link anucleophilic position on the ligand molecule to the amino function ofthe tether group on nucleoside sequence. By this mechanism, anoligonucleoside-maleimide conjugate is formed by reaction of the aminogroup of the tether on the linked nucleosides with the MBS or SMCCmaleimide linker. The conjugate is then reacted with the ligand.

Alternatively, a ligand conjugated-oligonucleotide can be prepared byconjugation of the ligand molecule to the oligonucleotide or nucleosidevia a homobifunctional tether such as disuccinimidyl suberate (DSS), tolink an amino function on the ligand to the amino group of a tether onthe oligonucleotide sequence. By this mechanism, anoligonucleoside-succinimidyl conjugate is formed by reaction of theamino group of the tether on the nucleoside sequence with adisuccinimidyl suberate tether. The disuccinimidyl suberate tethercouples with the amine tether on the nucleoside to extend the size ofthe tether. The extended tether is then reacted with an amino group ofthe ligand molecule.

Ligand-Bearing Nucleotides

The ligand-bearing nucleotides of the invention are pyrimidine-bearingnucleotides, wherein the C5-position of the pyrimidine base has beenmodified to form a covalent bond with the linker. In certain instances,the base of the pyrimidine-bearing nucleotide is a C5-modified thymine,cytosine, or uracil. As described above, the base, sugar, or phosphateportions of the nucleotide may be modified to incorporate unnaturalsubstituents.

A representative listing of C5-modified pyrimidine nucleosides andnucleotides amenable to the present invention are presented below.

Compound References

Bioorg. Med. Chem. Lett. 2002, 12(6), 981-983 U.S. Pat. No. 4,837,312 J.Am. Chem. Soc. 1989, 11 1(18), 6966-76.

Bioorg. Med. Chem. Lett. 2002, 12(6), 981-983 U.S. Pat. No. 4,837,312(06 Jun. 06, 1989) J. Am. Chem. Soc. 1989, 111(18), 6966-76 J. Org.Chem. 1991, 56(9), 3168-76

Bioorg. Med. Chem. Lett. 2002, 12(6), 981-983

J. Org. Chem. 1990, 55(17), 5125-32 U.S. Pat. No. 4,837,312, (06 Jun.06, 1989)

J. Org. Chem. 1990, 55(17), 5125-32

J. Org. Chem. 1990, 55(17), 5125-32

U.S. Pat. No. 4,837,312 (06 Jun. 06 1989)

U.S. Pat. No. 4,837,312 (06 Jun. 06 1989)

Ger. (East), 259803, 07 Sep. 1988

Ger. (East), 259803, 07 Sep. 1988

Ger. (East), 259803, 07 Sep. 1988

Ger. (East), 259803, 07 Sep. 1988

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Therapeutic Drug Monitoring 1992, 14(6), 499-508.Additional Features of Oligonuclotides

The siRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the siRNA agent, or a fragment thereof, can mediate down regulationof the target gene. It will be understood that the term “ribonucleotide”or “nucleotide” can, in the case of a modified RNA or nucleotidesurrogate, also refer to a modified nucleotide, or surrogate replacementmoiety at one or more positions. Thus, the siRNA agent is or includes aregion which is at least partially complementary to the target RNA. Incertain embodiments, the siRNA agent is fully complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the siRNA agent and the target, but the correspondence must besufficient to enable the siRNA agent, or a cleavage product thereof, todirect sequence specific silencing, such as by RNAi cleavage of thetarget RNA. Complementarity, or degree of homology with the targetstrand, is most critical in the antisense strand. While perfectcomplementarity, particularly in the antisense strand, is often desiredsome embodiments can include one or more but preferably 6, 5, 4, 3, 2,or fewer mismatches with respect to the target RNA. The mismatches aremost tolerated in the terminal regions, and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the over alldouble-strand character of the molecule.

In addition, a siRNA agent will often be modified or include nucleosidesurrogates. Single stranded regions of an siRNA agent will often bemodified or include nucleoside surrogates, e.g., the unpaired region orregions of a hairpin structure, e.g., a region which links twocomplementary regions, can have modifications or nucleoside surrogates.Modification to stabilize one or more 3′- or 5′-terminus of an iRNAagent, e.g., against exonucleases, or to favor the antisense siRNA agentto enter into RISC are also favored. Modifications can include C3 (orC6, C7, C12) amino linkers, thiol linkers, carboxyl linkers,non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol,hexaethylene glycol), special biotin or fluorescein reagents that comeas phosphoramidites and that have another DMT-protected hydroxyl group,allowing multiple couplings during RNA synthesis.

siRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed siRNA agents or shorter iRNA agentsherein. “siRNA agent or shorter siRNA agent” as used refers to an siRNAagent that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The siRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of a siRNA agent can be equal to or less than 30, 25, 24,23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred siRNA agents have a duplexregion of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, andone or more overhangs, preferably one or two 3′ overhangs, of 2-3nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an siRNA agent will preferably have one or more of thefollowing properties:

(1) it will, despite modifications, even to a very large number, or allof the nucleosides, have an antisense strand that can present bases (ormodified bases) in the proper three dimensional framework so as to beable to form correct base pairing and form a duplex structure with ahomologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(2) it will, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it willpossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, an siRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C_(3′)-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. Generally, it is preferred that a modified moiety at the2′ sugar position will be able to enter into H-bonding which is morecharacteristic of the OH moiety of a ribonucleotide than the H moiety ofa deoxyribonucleotide. A preferred siRNA agent will: exhibit aC_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of itssugars; exhibit a C_(3′)-endo pucker in a sufficient amount of itssugars that it can give rise to a the RNA-characteristic A-family-typehelix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which isnot a C_(3′)-endo pucker structure.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. A single strand iRNA agent should besufficiently long that it can enter the RISC and participate in RISCmediated cleavage of a target mRNA. A single strand iRNA agent is atleast 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

The compounds of the invention are described below in greater detail.Importantly, the embodiments described below are included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and are not intended to limit the invention.

One aspect of the present invention relates to a single-strandedoligonucleotide represented by formula I:

wherein

X is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂, or—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂;

M represents independently for each occurrence an alkali metal or atransition metal with an overall charge of +1;

n is 16, 17, 18, 19, 20, 21, 22, 23, or 24;

R¹, R², and R⁵ represent independently for each occurrence H, OH, F,—Oalkyl, —Oallyl, —O(C(R⁷)₂)_(v)OR⁷, —O(C(R⁷)₂)_(v)SR⁷,—O(C(R⁷)₂)_(v)N(R⁷)₂, —O(C(R⁷)₂)_(v)C(O)N(R⁷)₂, —N(R⁷)₂, —S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C₁-C₆)alkyl, —O(C(R⁷)₂)_(v)S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C(R⁷)₂)_(v)N((C₁-C₆)alkyl)₂, or—O(C(R⁷)₂)_(v)ON((C₁-C₆)alkyl)₂;

A¹ represents independently for each occurrence:

Z¹ represents independently for each occurrence O or S;

Z² represents independently for each occurrence —OH, —OM, —Oalkyl,—Oaryl, —Oaralkyl, —SH, —SM, —Salkyl, —Saryl, —Saralkyl, —N(R³)R⁴,—(C(R⁷)₂)_(m)N(R⁷)₂, —N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or alkyl;

R³ and R⁴ are H or alkyl; or R³ and R⁴ taken together form a 3-, 4-, 5-,6-, or 7-member ring;

R⁶ represents independently for each occurrence H, alkyl, or—NHCH₂CH═CH₂;

R⁷ represents independently for each occurrence H or alkyl;

A² represents independently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, aminoalkyl diradical, alkynyldiradical, alkylalkynyl diradical, thioether, —C(O)—, —S(O)—, —S(O)₂—,B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or has theformula:

B¹ represents independently for each occurrence a bond between A³ andA⁴;

B² represents independently for each occurrence a bond between A⁴ andA⁵;

R represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7 or 8;

m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7or 8;

v represents independently for each occurrence 1, 2, 3, or 4;

w represents independently for each occurrence 1, 2, or 3 in accord withthe rules of valence;

provided that A⁵ occurs at least once; and

A⁵ represents independently for each occurrence compound C, compound D,or a radical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12,riboflavin, biotin, aromatic compound, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring; wherein compound Cis represented by:

and compound C′ is represented by:

wherein for C and C′

R²⁵ represents independently for each occurrence H, halogen, hydroxyl,thiol, alkoxyl, thioalkyl, azido, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, —OC(O)R²⁷,alkyl, alkenyl, aryl, or aralkyl;

R²⁶ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl;

R²⁷ represents independently for, each occurrence H, alkyl, alkenyl,aryl, or aralkyl;

R²⁸ represents independently for each occurrence —OC(O)R²⁷,—N(R²⁷)C(O)R²⁷, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, alkyl, alkenyl, aryl, oraralkyl; and

p represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7;and

compound D is represented by:

and compound D′ is represented by:

wherein for D and D′

R²⁹ represents independently for each occurrence H, halogen, hydroxyl,thiol, alkoxyl, thioalkyl, azido, —N(R³⁰)₂, —CO₂R³⁰, —COR³⁰, —OC(O)R³⁰,alkyl, alkenyl, aryl, or aralkyl;

R³⁰ represents independently for each occurrence H, alkyl, alkenyl,aryl, or aralkyl;

R³¹ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl; and

t represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 18, 19, 20, 21, or 22.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 20.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least twice.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least five times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least ten times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence compound C, compound D, or a radical of cholesterol,5β-cholanic acid, progesterone, aldosterone, dehydroaldosterone,isoandrosterone, esterone, estradiol, ergosterol, dehydroergosterol,lanosterol, 4-cholesten-3-one, guggulsterone, testosterone,nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of cholesterol or 5β-cholanic acid.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence:

wherein R²⁰ represents independently for each occurrence hydroxyl,amino, halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, oracyloxy; Z³ represents independently for each occurrence O, S, or NR;and y represents independently for each occurrence 0, 1, 2, 3, 4, 5, or6 in accord with the rules of valence.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of cholic acid, deoxycholic acid, taurocholic acid,glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid,ursodeoxycholic acid, or chenodeoxycholic acid.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of lauric acid, myristic acid, palmitic acid,stearic acid, arachidic acid, palmitoleic acid, oleic acid, linoleicacid, linolenic acid, arachidonic acid, triacylglycerol,phosphoacylglycerol, sphingolipid, monoterpene, sesquiterpene,diterpene, sesterterpene, triterpene, or tetraterpene.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence optionally substituted phenyl, naphthyl, anthracenyl,phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl,phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl,quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl,benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl,pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl,tetrazolyl, or furanyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence the radical of erythrose, threose, ribose, arabinose, xylose,lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, ortalose; or a disaccharide or trisaccharide formed via a 1,4 glycosidelinkage between any of them.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is a hexose or pentose.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

wherein R⁴⁰ represents independently for each occurrence H, alkyl, aryl,aralkyl, —C(O)alkyl, —C(O)aryl, —C(O)aralkyl, or Si(alkyl)₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is an optionally substitutedcyclopentane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, or1,1-difluorocyclopentane.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound C.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound C′.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A5 is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound D.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound D′.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is a radical of a polycycliccompound.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

wherein R³² represents independently for each occurrence H, halogen,hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R³⁴)₂, —CO₂R³⁴, —COR³⁴,—OC(O)R³⁴, alkyl, alkenyl, aryl, or aralkyl; R³³ is aryl that isoptionally substituted with alkyl, alkenyl, aryl, halogen, or alkoxyl;and R³⁴ represents independently for each occurrence H, alkyl, aryl, oraralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² represents independently for eachoccurrence H, OH, F, or —Oalkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR⁷, —O(CH₂)₂SR⁷, —O(CH₂)₂N(R⁷)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² represents independently for eachoccurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR⁷, —O(CH₂)₂SR⁷, —O(CH₂)₂N(R⁷)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ represents independently for eachoccurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R⁵ represent independently foreach occurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence —OH, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SH, —SM, —Salkyl,—Saryl, —Saralkyl, —N(R³)R⁴, —(C(R⁷)₂)_(m)N(R⁷)₂,—N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or methyl;

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence —OH, —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned oligonucleotide, wherein A² represents independently foreach occurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ is a steroid.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence and A⁵ represents independently for each occurrence:

R²⁰ represents independently for each occurrence hydroxyl, amino,halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, or acyloxy;Z³ represents independently for each occurrence O, S, or NR; and yrepresents independently for each occurrence 0, 1, 2, 3, 4, 5, or 6 inaccord with the rules of valence.

Another aspect of the present invention relates to a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second strand are represented independentlyby formula II:

wherein

X is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂, or—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂;

M represents independently for each occurrence an alkali metal or atransition metal with an overall charge of +1;

n is 16, 17, 18, 19, 20, 21, 22, 23, or 24;

R¹, R², and R⁵ represent independently for each occurrence H, OH, F,—Oalkyl, —Oallyl, —O(C(R⁷)₂)_(v)OR⁷, —O(C(R⁷)₂)_(v)SR⁷,—O(C(R⁷)₂)_(v)N(R⁷)₂, —O(C(R⁷)₂)_(v)C(O)N(R⁷)₂, —N(R⁷)₂, —S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C₁-C₆)alkyl, —O(C(R⁷)₂)_(v)S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C(R⁷)₂)_(v)N((C₁-C₆)alkyl)₂, or—O(C(R⁷)₂)_(v)ON((C₁-C₆)alkyl)₂;

A¹ represents independently for each occurrence:

Z¹ represents independently for each occurrence O or S;

Z² represents independently for each occurrence —OH, —OM, —Oalkyl,—Oaryl, —Oaralkyl, —SH, —SM, —Salkyl, —Saryl, —Saralkyl, —N(R³)R⁴,—(C(R⁷)₂)_(m)N(R⁷)₂, —N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or alkyl;

R³ and R⁴ are H or alkyl; or R³ and R⁴ taken together form a 3-, 4-, 5-,6-, or 7-member ring;

R⁶ represents independently for each occurrence H, alkyl, or—NHCH₂CH═CH₂;

R⁷ represents independently for each occurrence H or alkyl;

A² represents independently for each occurrence:

A³ represents independently for each occurrence or

A⁴ represents independently for each occurrence a bond, alkyl diradical,heteroalkyl diradical, alkenyl diradical, aminoalkyl diradical, alkynyldiradical, alkylalkynyl diradical, thioether, —C(O)—, —S(O)—, —S(O)₂—,B¹C(R)₂B², B¹C(R)(B²)₂, B¹C(B²)₃, B¹N(R)(B²), B¹N(B²)₂, or has theformula:

B¹ represents independently for each occurrence a bond between A³ andA⁴;

B² represents independently for each occurrence a bond between A⁴ andA⁵;

R represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7 or 8;

m¹ represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7or 8;

v represents independently for each occurrence 1, 2, 3, or 4;

w represents independently for each occurrence 1, 2, or 3 in accord withthe rules of valence;

provided that A⁵ occurs at least once; and

A⁵ represents independently for each occurrence compound C, compound D,or a radical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12,riboflavin, biotin, aromatic compound, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring; wherein compound Cis represented by:

and compound C′ is represented by:

wherein for C and C′

R²⁵ represents independently for each occurrence H, halogen, hydroxyl,thiol, alkoxyl, thioalkyl, azido, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, —OC(O)R²⁷,alkyl, alkenyl, aryl, or aralkyl;

R²⁶ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl;

R²⁷ represents independently for each occurrence H, alkyl, alkenyl,aryl, or aralkyl;

R²⁸ represents independently for each occurrence —OC(O)R²⁷,—N(R²⁷)C(O)R²⁷, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, alkyl, alkenyl, aryl, oraralkyl; and

p represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7;and

compound D is represented by:

and compound D′ is represented by:

wherein for D and D′

R²⁹ represents independently for each occurrence H, halogen, hydroxyl,thiol, alkoxyl, thioalkyl, azido, —N(R³⁰)₂, —CO₂R³⁰, —COR³⁰, —OC(O)R³⁰,alkyl, alkenyl, aryl, or aralkyl;

R³⁰ represents independently for each occurrence H, alkyl, alkenyl,aryl, or aralkyl;

R³¹ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl; and

t represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 18, 19, 20, 21, or 22.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 20.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 20, and said first strand and saidsecond strand are hybridized so that there are two unhybridizednucleotides on said first strand and said second strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 20 for said first strand, and n is22 for said second strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least twice.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least five times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs at least ten times.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ occurs only in said first strand.

In certain embodiments, the present invention relates to theaforementioned compound, wherein said first strand and said secondstrand each contain at least one occurrence of A⁵.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence compound C, compound D, or a radical of cholesterol,5β-cholanic acid, progesterone, aldosterone, dehydroaldosterone,isoandrosterone, esterone, estradiol, ergosterol, dehydroergosterol,lanosterol, 4-cholesten-3-one, guggulsterone, testosterone,nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of cholesterol or 5β-cholanic acid.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence:

R²⁰ represents independently for each occurrence hydroxyl, amino,halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, or acyloxy;Z³ represents independently for each occurrence O, S, or NR; and yrepresents independently for each occurrence 0, 1, 2, 3, 4, 5, or 6 inaccord with the rules of valence.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of cholic acid, deoxycholic acid, taurocholic acid,glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid,ursodeoxycholic acid, or chenodeoxycholic acid.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence a radical of lauric acid, myristic acid, palmitic acid,stearic acid, arachidic acid, palmitoleic acid, oleic acid, linoleicacid, linolenic acid, arachidonic acid, triacylglycerol,phosphoacylglycerol, sphingolipid, monoterpene, sesquiterpene,diterpene, sesterterpene, triterpene, or tetraterpene.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence optionally substituted phenyl, naphthyl, anthracenyl,phenanthrenyl, pyrenyl, pyridinyl, quinolinyl, acridinyl,phenathridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinoxalinyl,quinazolinyl, 1,7-phenanthrolinyl, indolyl, thianaphthenyl,benzoxazolyl, benzofuranyl, 1,2-benzisoxazolyl, benzimidazolyl,pyrrolyl, thiophenyl, isoxazolyl, pyrazolyl, thiazolyl, imidazolyl,tetrazolyl, or furanyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ represents independently for eachoccurrence the radical of erythrose, threose, ribose, arabinose, xylose,lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, ortalose; or a disaccharide or trisaccharide formed via a 1,4 glycosidelinkage between any of them.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is a hexose or pentose.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

wherein R⁴⁰ represents independently for each occurrence H, alkyl, aryl,aralkyl, —C(O)alkyl, —C(O)aryl, —C(O)aralkyl, or Si(alkyl)₃.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A5 is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is an optionally substitutedcyclopentane, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, or1,1-difluorocyclopentane.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound C.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound C′.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound D.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is compound D′.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is a radical of a polycycliccompound.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A5 is

wherein R³² represents independently for each occurrence H, halogen,hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R³⁴)₂, —CO₂R³⁴, —COR³⁴,—OC(O)R³⁴, alkyl, alkenyl, aryl, or aralkyl; R³³ is aryl that isoptionally substituted with alkyl, alkenyl, aryl, halogen, or alkoxyl;and R³⁴ represents independently for each occurrence H, alkyl, aryl, oraralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁵ is

and A⁴ is

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² represents independently for eachoccurrence H, OH, F, or —Oalkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR⁷, —O(CH₂)₂SR⁷, —O(CH₂)₂N(R⁷)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² represents independently for eachoccurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ represents independently for eachoccurrence H, OH, F, —OCH₃, —O(CH₂)₂OR⁷, —O(CH₂)₂SR⁷, —O(CH₂)₂N(R⁷)₂,—OCH₂C(O)N(H)CH₃, —NH₂, —N(CH₃)₂, —N(H)CH₃, —SCH₃, —O(CH₂)₂OCH₃,—O(CH₂)₂SCH₃, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, or —O(CH₂)₂ON(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁵ represents independently for eachoccurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² and R⁵ represent independently foreach occurrence —NH₂, —N(H)CH₃, or —N(CH₃)₂.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence —OH, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SH, —SM, —Salkyl,—Saryl, —Saralkyl, —N(R³)R⁴, —(C(R⁷)₂)_(m)N(R⁷)₂,—N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or methyl;

In certain embodiments, the present invention relates to theaforementioned compound, wherein Z² represents independently for eachoccurrence —OH, —OM, —Oalkyl, —Oaryl, or —Oaralkyl.

In certain embodiments, the present invention relates to theaforementioned oligonucleotide, wherein A² represents independently foreach occurrence:

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

In certain embodiments, the present invention relates to theaforementioned compound, wherein A⁴ represents independently for eachoccurrence

and A⁵ is a steroid.

In certain embodiments, the present invention relates to theaforementioned compound,

wherein A⁴ represents independently for each occurrence and A⁵represents independently for each occurrence:

R²⁰ represents independently for each occurrence hydroxyl, amino,halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, or acyloxy;Z³ represents independently for each occurrence O, S, or NR; and yrepresents independently for each occurrence 0, 1, 2, 3, 4, 5, or 6 inaccord with the rules of valence.Methods of the Invention

One aspect of the present invention relates to a method of treating apatient suffering from a malady selected from the group consisting ofunwanted cell proliferation, arthritis, retinal neovascularization,viral infection, bacterial infection, amoebic infection, parasiticinfection, fungal infection, unwanted immune response, asthma, lupus,multiple sclerosis, diabetes, acute pain, chronic pain, neurologicaldisease, and a disorder characterized by loss of heterozygosity;comprising the step of:

administering to a patient in need thereof a therapeutically effectiveamount of an oligonucleotide, wherein said oligonucleotide is asingle-stranded oligonucleotide represented by formula I as describedabove, or said oligonucleotide is a double-stranded oligonucleotidecomprising a first strand and a second strand, wherein said first strandand said second are represented independently by formula II as describedabove.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is unwanted cellproliferation.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is testicular cancer, lungcancer, breast cancer, colon cancer, squamous cell carcinoma, pancreaticcancer, leukemia, melanoma, Burkitt's lymphoma, neuroblastoma, ovariancancer, prostate cancer, skin cancer, non-Hodgkin lymphoma, esophagealcancer, cervical cancer, basal cell carcinoma, adenocarcinoma carcinoma,hepatocellular carcinoma, colorectal adenocarcinoma, liver cancer, malebreast carcinoma, adenocarcinomas of the esophagus, adenocarcinomas ofthe stomach, adenocarcinomas of the colon, adenocarcinomas of therectum, gall bladder cancer, hamartomas, gliomas, endometrial cancer,acute leukemia, chronic leukemia, childhood acute leukemia, EwingSarcoma, Myxoid liposarcoma, brain cancer, or tumors of epithelialorigin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is rheumatoid arthritis orretinal neovascularization.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a viral infection.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder mediated byHuman Papilloma Virus, Human Immunodeficiency Virus, Hepatitis A Virus,Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis EVirus, Hepatitis F Virus, Hepatitis G Virus, Hepatitis H Virus,Respiratory Syncytial Virus, Herpes Simplex Virus, herpesCytomegalovirus, herpes Epstein Barr Virus, a Kaposi'sSarcoma-associated Herpes Virus, JC Virus, myxovirus, rhinovirus,coronavirus, West Nile Virus, St. Louis Encephalitis, Tick-borneencephalitis virus gene, Murray Valley encephalitis virus gene, denguevirus gene, Simian Virus 40, Human T Cell Lymphotropic Virus, aMoloney-Murine Leukemia Virus, encephalomyocarditis virus, measlesvirus, Vericella zoster virus, adenovirus, yellow fever virus,poliovirus, or poxvirus.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a bacterial infection,amoebic infection, parasitic infection, or fungal infection.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder mediated byplasmodium, Mycobacterium ulcerans, Mycobacterium tuberculosis,Mycobacterium leprae, Staphylococcus aureus, Streptococcus pneumoniae,Streptococcus pyogenes, Chlamydia pneumoniae, or Mycoplasma pneumoniae.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is an unwanted immuneresponse, asthma, lupus, multiple sclerosis, or diabetes.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is an ischemia, reperfusioninjury, response to a transplantated organ or tissue, restenosis, orInflammatory Bowel Disease.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is acute pain or chronicpain.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a neurological disease.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is Alzheimer Disease,Parkinson Disease, or a neurodegenerative trinucleotide repeat disorder.

In certain embodiments, the present invention relates to theaforementioned method, wherein said malady is a disorder characterizedby loss of heterozygosity.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammalian cell to silence a gene promoting unwanted cellproliferation, growth factor gene, growth factor receptor gene, a kinasegene, a gene encoding a G protein superfamily molecule, a gene encodinga transcription factor, a gene which mediates angiogenesis, a viral geneof a cellular gene which mediates viral function, a gene of a bacterialpathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen,a gene of a fungal pathogen, a gene which mediates an unwanted immuneresponse, a gene which mediates the processing of pain, a gene whichmediates a neurological disease, an allene gene found in cellscharacterized by loss of heterozygosity, or one allege gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula I as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammalian cell to silence a PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Dgene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene,mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusion gene, BCR/ABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Flt-1 receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human Immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis EVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula I as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammal to silence a gene promoting unwanted cell proliferation,growth factor or growth factor receptor gene, a kinase gene, a geneencoding a G protein superfamily molecule, a gene encoding atranscription factor, a gene which mediates angiogenesis, a viral geneof a cellular gene which mediates viral function, a gene of a bacterialpathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen,a gene of a fungal pathogen, a gene which mediates an unwanted immuneresponse, a gene which mediates the processing of pain, a gene whichmediates a neurological disease, an allene gene found in cellscharacterized by loss of heterozygosity, or one allege gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula I as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Another aspect of the present invention relates to a method ofgene-silencing, comprising the steps of:

administering a therapeutically effective amount of an oligonucleotideto a mammal to silence a PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin D gene,VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21(WAF1/CIP1) gene,mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusion gene, BCR/ABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Flt-1 receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human Immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis EVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene; wherein said oligonucleotide is a single-strandedoligonucleotide represented by formula I as described above, or saidoligonucleotide is a double-stranded oligonucleotide comprising a firststrand and a second strand represented by formula II as described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein, said mammal is a primate, equine, canineor feline.

In certain embodiments, the present invention relates to theaforementioned method, wherein, said mammal is a human.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is a double-strandedoligonucleotide comprising a first strand and a second strand, whereinsaid first strand and said second are represented independently byformula II as described above.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “silence” means to at least partially suppress. For example, incertain instances, the gene is suppressed by at least about 25%, 35%, or50% by administration of the double stranded oligonucleotide of theinvention. In a preferred embodiment, the gene is suppressed by at leastabout 60%, 70%, or 80% by administration of the double strandedoligonucleotide of the invention. In a more preferred embodiment, thegene is suppressed by at least about 85%, 90%, or 95% by administrationof the double stranded oligonucleotide of the invention. In a mostpreferred embodiment, the gene is suppressed by at least about 98% or99% by administration of the double stranded oligonucleotide of theinvention.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as analgesics), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound in binding to sigmareceptors. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. Unless specified otherwise, thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and 1)ara are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” areart-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to—SO₂—. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on page 560 of “AdvancedInorganic Chemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In other embodiments, R50 and R51(and optionally R52) each independently represent a hydrogen, an alkyl,an alkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carboxyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 andR56 represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61 or apharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the formula representsan “ester”. Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the formula represents a “carboxylic acid”. Where X50is an oxygen, and R56 is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the formula represents a“thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the formula represents a “thiolformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above formula represents an “aldehyde” group.

The term “carbamoyl” refers to —O(C═O)NRR′, where R and R′ areindependently H, aliphatic groups, aryl groups or heteroaryl groups.

The term “oxo” refers to a carbonyl oxygen (═O).

The terms “oxime” and “oxime ether” are art-recognized and refer tomoieties that may be represented by the general formula:

wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61. The moiety is an “oxime” when R is H; and itis an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R58 is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The term “selenoalkyl” is art-recognized and refers to an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R61, m andR61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991). Protected forms of the inventive compounds are included withinthe scope of this invention.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; or (8) nasally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhybrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhybrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhybrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

Micelles

Recently, the pharmaceutical industry introduced microemulsificationtechnology to improve bioavailability of some lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other things, microemulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom a compound of the present invention and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Particularly preferred amphiphilic carriers are saturated andmonounsaturated polyethyleneglycolyzed fatty acid glycerides, such asthose obtained from fully or partially hydrogenated various vegetableoils. Such oils may advantageously consist of tri-. di- and mono-fattyacid glycerides and di- and mono-polyethyleneglycol esters of thecorresponding fatty acids, with a particularly preferred fatty acidcomposition including capric acid 4-10, capric acid 3-9, lauric acid40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%.Another useful class of amphiphilic carriers includes partiallyesterified sorbitan and/or sorbitol, with saturated or mono-unsaturatedfatty acids (SPAN-series) or corresponding ethoxylated analogs(TWEEN-series).

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Polymers

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhybrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter .alpha., .beta. or.gamma., respectively. Cyclodextrins with fewer than six glucose unitsare not known to exist. The glucose units are linked byalpha-1,4-glucosidic bonds. As a consequence of the chair conformationof the sugar units, all secondary hydroxyl groups (at C-2, C-3) arelocated on one side of the ring, while all the primary hydroxyl groupsat C-6 are situated on the other side. As a result, the external facesare hydrophillic, making the cyclodextrins water-soluble. In contrast,the cavities of the cyclodextrins are hydrophobic, since they are linedby the hydrogen of atoms C-3 and C-5, and by ether-like oxygens. Thesematrices allow complexation with a variety of relatively hydrophobiccompounds, including, for instance, steroid compounds such as17.beta.-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org.Cult. 38:1-3-113 (1994)). The complexation takes place by Van der Waalsinteractions and by hydrogen bond formation. For a general review of thechemistry of cyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl.,33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μmOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C 14 to about C 20). Polymer-derivatized lipidssuch as PEG-lipids may also be utilized for micelle formation as theywill act to inhibit micelle/membrane fusion, and as the addition of apolymer to surfactant molecules decreases the CMC of the surfactant andaids in micelle formation. Preferred are surfactants with CMCs in themicromolar range; higher CMC surfactants may be utilized to preparemicelles entrapped within liposomes of the present invention, however,micelle surfactant monomers could affect liposome bilayer stability andwould be a factor in designing a liposome of a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

Release Modifiers

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Procedure for Preparation of Nucleoside-Ligand ConjugatesContaining Uridine Analogues

(i) For 2a: cholesterylchloroformate, TEA/dichloromethane; for 2b:5β-cholanic acid pentafluorophenol ester, TEA/dichloromethane; (ii) (a)succinic anhybride, DMAP/dichloroethane and (b) DTNP, DMAP, Ph₃P,aminoalkyl solid support; (iii) N,N-diisopropylaminoβ-cyanoethylphosphonamidic chloride {[(CH₃)₂CH]₂N—P(Cl)—OCH₂CH₂CN},DIEA/dichloromethane or 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane,tetrazole (or tetrazolediisopropylammonium salt)/acetonitrile.

Preparation of Compound 51a:

DMT-dT-C5-Amino linker (50, 1.00 g, 1.43 mmol) from Chem Genes wasstirred with cholesteryl chloroformate (0.77 g, 1.71 mmol) indichloromethane (10 mL) in the presence of TEA (1.0 mL) at ambienttemperature for 2 h. Completion of the reaction was confirmed by TCLmonitoring. The reaction mixture was diluted to 50 mL by adding moredichloromethane and washed successively with NaHCO₃ solution and waterfollowed by standard workup. Residue obtained was purified by flashsilica gel column chromatography to afford 51a (0.66 g, 37.75%). ¹H NMR(400 MHz, [D₆]DMSO, 25° C.): δ 11.61 (s, 1H, exchangeable with D₂-O),8.01-7.98 (t, J(H,H)=5.39 Hz, 1H, exchangeable with D₂O), 7.92 (s, 1H),7.37-6.99 (m, 12H), 6.87-6.83 (m, 4H), 6.17-6.14 (t, J(H,H)=6.64 Hz,1H), 5.30 (s, 1H), 5.28-5.27 (d, J(H,H)=4.56 Hz, 1H, exchangeable withD₂O), 4.32-4.20 (m, 2H), 3.87-3.84 (m, 1H), 3.71-3.63 (m, 7H), 3.21-3.03(m, 4H), 2.95-2.88 (m, 2H), 2.33-2.13 (m, 4H), 1.97-1.73 (m, 5H),1.54-0.82 (m, 40H), 0.63 (s, 3H).

Preparation of Compound 52a:

Compound 51a (0.55 g, 0.495 mmol) and succinic anhybride (0.075 g, 0.749mmol) were suspended in anhydrous dichloromethane (5 mL) and stirred atambient temperature in the presence of DMAP (0.18 g, 1.49 mmol)overnight. After confirming completion of the reaction, the reactionmixture was diluted to 50 mL by adding dichloromethane and washed withdilute citric acid solution; organic layer was dried over anhydrousNa₂SO₄ and evaporated in vacuo. Residue obtained was purified by flashsilica gel column chromatography, eluent 6% methanol in dichloromethane,to afford the corresponding succinic acid derivative (0.50 g, 83.4%). ¹HNMR (400 MHz, [D₆]DMSO, 25° C.): δ 12.24 (bs, 1H, exchangeable withD₂O), 11.64 (s, 1H, exchangeable with D₂O), 8.02-7.99 (bm, 2H),7.36-7.00 (m, 12H), 6.87-6.81 (m, 4H), 6.15-6.11 (t, J(H,H)=6.84 Hz,1H), 5.30 (bs, 1H), 5.17-5.14 (bm, 1H), 4.31-4.24 (m, 1H), 4.05 (bm,1H), 3.70-3.66 (m, 8H), 3.34-3.08 (m, 6H), 2.94-2.88 (m, 4H), 2.31-2.13(m, 3H), 1.96-1.71 (bm, 5H), 1.55-0.80 (m, 40H), 0.63 (s, 3H).

The succinnate thus obtained was conjugated to long chain aminoalkylcontrolled glass support (CPG) with a loading of 155 μM/g loading asdescribed in the literature by Kumar et al. (Nucleosides andNucleotides, 1996, 15, 879) to obtain the desired the desired CPG solidsupport 52a (1.70 g) with a loading of 78.42 μM/g. The loading of thesupport 3a was determined as described in the literature (Prakash etal., J. Org. Chem., 2002, 67, 357).

Preparation of Compound 53a:

The phosphoramidite 53a is prepared from compound 51a by reacting with2-Cyanoethyl-N,N,N,N′-tetraisopropylphosphane in the presence oftetrazolediisopropylammonium salt in acetonitrile according to reportedprocedures (Rajeev et al., Org. Lett., 2003, 5, 3005).

Preparation of Compound 51b:

5β-Cholanic acid (5.00 g, 13.87 mmol, purchased from Sigma),pentafluorophenol (2.81 g, 15.27 mmol, purchased from Aldrich) and DMAP(0.20 g, 1.64 mmol) were dissolved in dichloromethane andN,N′-dicyclohexycarbodiimide (DCC, 2.86 g, 13.86 mmol) was added intothe solution at ambient temperature. The reaction mixture was stirredfor 4 h. N,N′-Dicyclohexylurea was filtered off from the reaction andthe filtrate was evaporated to obtain pentafluorophenol ester of5β-cholanic acid. The ester (0.90 g, 1.708 mmol) thus obtained wasstirred with compound 50 (1.00 g, 1.431 mmol) in the presence of TEA indichloromethane (8 mL) for 2 h. The reaction was complete after 2 h asevident from TLC analysis. Reaction mixture was diluted to 50 mL byadding more dichloromethane and washed with dilute NaHCO₃ solutionfollowed by standard workup. Residue was purified by flash silica gelcolumn chromatography, eluent 3-4% methanol in dichloromethane, toafford the desired compound 51b (1.46 g, 98.04%). ¹H NMR (400 MHz,[D₆]DMSO, 25° C.): δ 11.62 (bs, 1H exchangeable with D₂O); 8.03-8.00 (t,J(H,H)=5.38 Hz, 1H, exchangeable with D₂O), 7.92 (s, 1H), 7.74-7.71 (t,1H, exchangeable with D₂O); 7.37-7.02 (m, 11H), 6.88-6.84 (m, 4H),6.17-6.14 (t, J(H.H)=6.35, 6.69 Hz, 1H), 4.22-4.19 (m, 1H), 3.88-3.85(m, 1H), 3.70-3.69 (d, J(H,H)=3.91 Hz, 6H); 3.20-2.89 (m, 6H), 2.33-2.27(m, 1H), 2.18-2.12 (m, 1H), 2-08-2.00 (m, 1H), 1.99-1.84 (m, 2H),1.84-1.56 (m, 6H), 1.54-0.94 (m, 33H), 0.87-0.79 (m, 7H), 0.57 (s, 3H).¹³C NMR (100 MHz, [D₆]DMSO, 25° C.): δ 173.5, 166.1, 162.2, 158.5,149.7, 145.2, 142.9, 136.0, 135.9, 132.5, 130.1, 128.4, 128.1, 127.2,122.4, 113.6, 109.8, 107.3, 86.1, 85.9, 85.4, 70.6, 64.2, 56.5, 56.0,55.4, 55.2, 46.2, 43.5, 42.7, 40.4, 38.7, 37.5, 35.8, 35.3, 32.9, 32.1,29.5, 29.4, 28.2, 27.5, 27.1, 26.9, 26.6, 26.5, 24.4, 24.3, 21.2, 20.9,18.6, 12.2, 9.1.

Preparation of Compound 52b:

Compound 52b was prepared from compound 51b as described above for thesynthesis of compound 52a. Loading of the support 52b (2.7 g) preparedwas determined as 81 μM/g.

Preparation of Compound 53b:

The phosphoramidite 53b is prepared from compound 51b by reacting with2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphane in the presence oftetrazolediisopropylammonium salt in acetonitrile according to reportedprocedures (Rajeev et al., Org. Lett., 2003, 5, 3005).

Example 2

The siRNA duplexes to be used for biological assays are listed below inTable I.

TABLE I siRNA Duplexes Containing Lipophilic Cholesterol or 5β-CholanicAcid at the C5-Position of the Pyrimidine (SEQ ID NOS: 2-93,respectively, in order of appearance) Number Name Sequence^(a) 101 Luc^(5′)CUUACGCUGAGUACUUCGAdTdT^(3′) ^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 102Luc-cholesterol 3′-end ^(5′)CUUACGCUGAGUACUUCGAdTdt^(3′)^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 103 Luc-cholesterol 5′-end^(5′)dcUUACGCUGAGUACUUCGAdTdT^(3′) ^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 104Luc-5β-cholanic acid ^(5′)CUUACGCUGAGUACUUCGAdTdt ^(3′) 3′-end^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 105 Luc-5β-cholanic acid^(5′)dcUUACGCUGAGUACUUCGAdTdT^(′) 5′-end^(3′)dTdTGAAUGCGACUCAUGAAGCU^(5′) 106 Luc Scrambled^(5′)CAUUCUCGGUGACAUUCGAdTdT^(3′) ^(3′)dTdTGUAAGAGCCACUGUAAGCU^(5′) 107Luc - cholesterol 3′- ^(5′)CAUUCUCGGUGACAUUCGAdTdt^(3′) end Scrambled^(3′)dTdTGUAAGAGCCACUGUAAGCU^(5′) 108 Luc - cholesterol 5′-^(3′)dcAUUCUCGGUGACAUUCGAdTdT^(3′) end Scrambled^(3′)dTdTGUAAGAGCCACUGUAAGCU^(5′) 109 Luc - 5β-cholanic acid^(5′)CAUUCUCGGUGACAUUCGAdTdt ^(3′) 3′-end Scrambled^(3′)dTdTGUAAGAGCCACUGUAAGCU^(5′) 110 Luc - 5β-cholanic acid^(5′)dcAUUCUCGGUGACAUUCGAdTdT^(3′) 5′-end Scrambled^(3′)dTdTGUAAGAGCCACUGUAAGCU^(5′) 111 VEGF^(5′)GCGGAUCAAACCUCACCAAdTdT^(3′) ^(3′)dTdTCGCCUAGUUUGGAGUGGUU^(5′) 112VEGF - cholesterol ^(5′)GCGGAUCAAACCUCACCAAdTdt^(3′)^(3′)dTdTCGCCUAGUUUGGAGUGGUU^(5′) 113 VEGF - 5β-cholanic acid^(5′)GCGGAUCAACCUCACCAAdTdt ^(3′) ^(3′)dTdTCGCCUAGUUUGGAGUGGUU^(5′) 114VEGF Scrambled ^(5′)GCGGAACAAUCCUGACCAAdTdT^(3′)^(3′)dTdTCGCCUUGUUAGGACUGGUU^(5′) 115 VEGF - cholesterol^(5′)GCGGAACAAUCCUGACCAAdTdt^(3′) Scrambled^(3′)dTdTCGCCUUGUUAGGACUGGUU^(5′) 116 VEGF - 5β-cholanic acid^(5′)GCGGAACAAUCCUGACCAAdTdt ^(3′) Scrambled^(3′)dTdTCGCCUUGUUAGGACUGGUU^(5′) 117 PTEN^(b)^(5′)CAAAUCCAGAGGCUAGCAGdTdT^(3′) ^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 118PTEN-cholesterol 3′-end ^(5′)CAAAUCCAGAGGCUAGCAGdTdt^(3′)^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 119 PTEN-cholesterol 5′-end^(5′)dcAAAUCCAGAGGCUAGCAGdTdT ^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 120PTEN-scramble ^(5′)CAACGAGCGAACUGCGAAUdTdT^(3′)^(3′)dTdTGUUGCUCGCUUGACGCUUA^(5′) 121 PTEN-cholesterol -^(5′)CAACGAGCGAACUGCGAAUdTdt^(3′) scramble 3′-end^(3′)dTdTGUUGCUCGCUUGACGCUUA^(5′) 122 PTEN - cholesterol^(5′)dcAACGAGCGAACUGCGAAUdTdT^(3′) scramble 5′-end^(3′)dTdTGUUGCUCGCUUGACGCUUA^(5′) 123 PTEN - 5β-cholanic acid^(5′)CAAAUCCAGAGGCUAGCAGdTdt ^(3′) 3′- end^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 124 PTEN - 5β-cholanic acid ^(5′)dcAAAUCCAGAGGCUAGCAGdTdT end ^(3′)dTdTGUUUAGGUCUCCGAUCGUC^(5′) 125PTEN - 5β-cholanic acid ^(5′)CAACGAGCGAACUGCGAAUdTdt ^(3′) scramble3′-end ^(3′)dTdTGUUGCUCGCUUGACGCUUA^(5′) 126 PTEN - 5β-cholanic acid^(5′)dcAACGAGCGAACUGCGAAUdTdT^(3′) scramble 5′-end^(3′)dTdTGUUGCUCGCUUGACGCUUA^(5′) 127 c - raf^(c)^(5′)AUGCAUGUCACAGGCGGGAdTdT^(3′) ^(3′)dTdTUACGUACAGUGUCCGCCCU^(5′) 128c-raf-cholesterol ^(5′)AUGCAUGUCACAGGCGGGAdTdt^(3′)^(3′)dTdTUACGUACAGUGUCCGCCCU^(5′) 129 c-raf-5β-cholanic acid^(5′)AUGCAUGUCACAGGCGGGAdTdt ^(3′) ^(3′)dTdTUACGUACAGUGUCCGCCCU^(5′) 130c-raf-scramble ^(5′)GUAGCAGCGGACAGAGUCUdTdT^(3′)^(3′)dTdTCAUCGUCGCCUGUCUCAGA^(5′) 131 c-raf -cholesterol^(5′)GUAGCAGCGGACAGAGUCUdTdt^(3′) scramble^(3′)dTdTCAUCGUCGCCUGUCUCAGA^(5′) 132 c-raf-5β-cholanic acid^(5′)GUAGCAGCGGACAGAGUCUdTdt ^(3′) scramble^(3′)dTdTCAUCGUCGCCUGUCUCAGA^(5′) 133 H-ras^(d)^(5′)CCGUGAGGAGAGAUGACGGdTdT^(3′) ^(3′)dTdTGGGACUCCUCGCUACUGCC^(5′) 134H-ras- cholesterol 3′- ^(5′)CCGUGAGGAGAGAUGACGGdTdt^(3′) end^(3′)dTdTGGGACUCCUCGCUACUGCC^(5′) 135 H-ras- cholesterol 5′-^(5′)dcCGUGAGGAGAGAUGACGGdTdT^(3′) end ^(3′)dTdTGGGACUCCUCGCUACUGCC^(5′)136 H-ras- 5β-cholanic acid ^(5′)CCGUGAGGAGAGAUGACGGdTdt ^(3′) 3′-end^(3′)dTdTGGGACUCCUCGCUACUGCC^(5′) 137 H-ras- 5β-cholanic acid ^(5′)dcCGUGAGGAGAGAUGACGGdTdT^(3′) 5′-end ^(3′)dTdTGGGACUCCUCGCUACUGCC^(5′)138 H-ras-scramble ^(5′)CGCGAAGAAGUGCGUGAGGdTdT^(3′)^(3′)dTdTGCGCUUCUUCACGCACUCC^(5′) 139 H-ras -cholesterol -^(5′)CGCGAAGAAGUGCGUGAGGdTdt^(3′) scramble 3′-end^(3′)dTdTGCGCUUCUUCACGCACUCC^(5′) 140 H-ras-5β-cholanic acid^(5′)CGCGAAGAAGUGCGUGAGGdTdt ^(3′) -scramble 3′-end^(3′)dTdTGCGCUUCUUCACGCACUCC^(5′) 141 Apo-B^(e)^(5′)AAGGUGUAUGGCUUCAACCCUdTdT^(3′) ^(3′)dTdTUUCCACAUACCGAAGUUGGGA^(5′)142 Apo-B - cholesterol ^(5′)AAGGUGUAUGGCUUCAACCCUdTdt^(3′)^(3′)dTdTUUCCACAUACCGAAGUUGGGA^(5′) 143 Apo-B - 5β-cholanic^(5′)AAGGUGUAUGGCUUCAACCCUdTdt-^(3′) acid^(3′)dTdTUUCCACAUACCGAAGUUGGGA^(5′) 144 Apo-B-scramble^(5′)UGCCACUCUGAGAAUAGGCUUdTdT^(3′) ^(3′)dTdTACGGUGAGACUCUUAUCCGAA^(5′)145 Apo-B-cholesterol- ^(5′)UGCCACUCUGAGAAUAGGCUUdTdt^(3′) scramble^(3′)dTdTACGGUGAGACUCUUAUCCGAA^(5′) 146 Apo-B-5β-cholanic acid^(5′)UGCCACUCUGAGAAUAGGCUUdTdt ^(3′) - scramble^(3′)dTdTACGGUGAGACUCUUAUCCGAA^(5′) ^(a)The sense strand is written5′ to 3′ on the top line. The antisense strand is written 3′ to5′ below. The oligonucleotides are phosphodiester RNA except for two3′ deoxythymidines indicated by dT in the sequence. dt representcholesterol conjugation at C5 of 2′-deoxyuridine and dc representcholesterol conjugation at C5 of 2′-deoxycytidine. dt represent5β-cholanic acidconjugation at C5 of 2′-deoxyuridine and dc represent5β-cholanic acid conjugation at C5 of 2′- deoxycytidine. Scrambledsequences were generated by randomizing the sequence of the sensestrand. ^(b)The PTEN sequence is identical (with the exception of the3′dTdT) on the antisense strand to that of an antisense oligonucleotidewith pharmacological activity. [1] ^(c)The c-raf sequence is identical(with the exception of the 3′dTdT) on the antisense strand to that of anantisense oligonucleotide with pharmacological activity. [2] ^(d)TheH-ras sequence is identical (with the exception of the 3′dTdT) on theantisense strand to that of an antisense oligonucleotide withpharmacological activity. [3] ^(e)The Apo-B sequence is identical (withthe exception of the 3′dTdT) on the antisense strand to that of anantisense oligonucleotide with pharmacological activity. [3]

REFERENCES

-   [1] M. Butler, R. A. McKay, I. J. Popoff, W. A. Gaarde, D.    Witchell, S. F. Murray, N. M. Dean, S. Bhanot, B. P. Monia,    Diabetes. 2002 51, 1028.-   [2] B. P. Monia, H. Sasmor, J. F. Johnston, S. M. Freier, E. A.    Lesnik, M. Muller, T. Geiger, K. H. Altmann, H. Moser, D., Proc.    Natl. Acad. Sci., USA 1996 93, 15481.-   [3] L. M. Cowsert, Anti-Cancer Drug Design 1997 12, 359.-   [4] R. M. Crooke, M. J. Graham, PCT Int. Appl. (2003), WO 2003097662    A1 20031127

Example 3 Oligonucleotides: Synthesis, Purification and Analysis

Synthesis:

The designed RNA molecules are synthesized on a 394 ABI machine usingthe standard 93 step cycle written by the manufacturer withmodifications to a few wait steps as described below. Solid supports 52aand 52b and phosphoramidites 53a and 53b are used for the synthesis ofdesired oligonucleotides along with commercially available DNA and RNAphosphoramidites. Commercial phosphoramidites with fast protectinggroups (5′-O-dimethoxytritylN6-phenoxyacetyl-2′-O-t-butyldimethylsilyladenosine-3′-O—N,N′-diisopropyl-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-4-acetyl-2′-O-t-butyldimethylsilylcytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-2-p-isopropylphenoxyacetyl-2′-O-t-butyldimethylsilylguanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyluridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditeare purchased either from Pierce Nucleic Acids Technologies, Milwaukee,Wis. or from Proligo LLC, Boulder, Colo. All 2′-O-Me amidites arereceived from Glen Research. All amidites are used at a concentration of0.15 M in acetonitrile (CH₃CN) and a coupling time of 8-15 min. Theactivator is 5-(ethylthio)-1H-tetrazole (0.25 M), for the PO-oxidationIodine/Water/Pyridine is used and for PS-oxidation, 2% Beaucage reagent(Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253) in anhydrousacetonitrile is used. The sulphurization time is about 6 min.

Deprotection-I (Nucleobase Deprotection):

After completion of synthesis the support is transferred to a screw capvial (VWR Cat # 20170-229) or screw cap RNase free microfuge tube. Theoligonucleotide is cleaved from the support with simultaneousdeprotection of base and phosphate groups with 1.0 mL of a mixture ofethanolic ammonia [ammonia: ethanol (3:1)] for 15 h at 55° C. The vialis cooled briefly on ice and then the ethanolic ammonia mixture istransferred to a new microfuge tube. The CPG is washed with 2×0.1 mLportions of RNase free deionised water. Combined washings, cools over adry ice bath for 10 min and subsequently dries in speed vac.

Deprotection-II (Removal of 2′ TBDMS Group):

The white residue obtains is resuspended in 400 II of triethylamine,triethylamine trihydrofluoride (TEA.3HF) and NMP (4:3:7) and heats at50° C. for overnight to remove the tert-butyldimethylsilyl (TBDMS)groups at the 2′position (Wincott et al., Nucleic Acids Res., 1995, 23,2677). The reaction is then quenched with 400 μl ofisopropoxytrimethylsilane (iPrOMe₃Si, purchased from Aldrich) andfurther incubates on the heating block leaving the caps open for 10 min;(This causes the volatile isopropxytrimethylsilylfluoride adduct tovaporize). The residual quenching reagent is removed by drying in aspeed vac. 1.5 ml of 3% triethylamine in diethyl ether is added and theoligonucleotide is pelleted out by centrifuging. The supernatant ispipetted out without disturbing the pellet and the pellet is dried inspeed vac to obtain the crude oligonucleotide as a white fluffymaterial.

Quantitation of Crude Oligomer or Raw Analysis:

Samples are dissolved in RNase free deionied water (1.0 mL) andquantitates as follows: Blanking is first performed with water alone (1mL); 20 μL of sample and 980 μL of water are mixed well in a microfugetube, transfers to cuvette and absorbance reading is obtained at 260 nm.The crude material is dried down and stored at −20° C.

Purification of Oligomers:

PAGE Purification

PAGE purification of oligomer synthesized is performed as reported bySambrook et al. (Molecular Cloning: a Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). A12% denaturing gel is prepared for purification of unmodified andmodified oligonucleotides. To a mixture of 120 mL Concentrate, 105 mLDiluents and 25 mL Buffer (National Diagnostics) is added 50 μL TEMEDand 1.5 mL 10% APS. After pouring the gel, it is left for ½ h topolymerize. Oligonucleotide is suspended in 20 μL water and 80 μLformamide. Loads gel tracking dye on left lane followed by the sampleslowly on to the gel. Run the gel on 1×TBE buffer at 36 W for 4-6 h.Once run is completed, transfer the gel on to preparative TLC plates andsee under UV light. Cut the bands, soak and crush in RNase free waterand leaves the vial containing purified oligonucletide in a shaker forovernight. Eluent is removed, wash residue with more RNase free water,combined washing and lyophilize to obtain the pure oligonucleotide.

Desalting of Purified Oligomer:

The purified dry oligomer is desalted using Sephadex G-25 M (AmershamBiosciences). The cartridge is conditioned with 10 mL of RNase freedeionised water thrice. Finally the purified oligomer is dissolved in2.5 mL RNasefree water and passed through the cartridge with very slowdrop wise elution. The salt free oligomer is eluted with 3.5 mL of RNasefree water directly into a screw cap vial.

Analysis:

Capillary Gel Electrophoresis (CGE) and Electrospray LC/MS

Approximately 0.10 OD of oligomer is first dried down, then redissolvsin water (50 μL) and pipettes in specified vials for CGE and LC/MSanalysis.

Example 4 In Vitro Cell Culture Activities of Ligand Conjugated siRNADuplex

Dual Luciferase Gene Silencing Assays:

In vitro activity of siRNAs is determined using a high-throughput96-well plate format luciferase silencing assay. Assays are performed inone of two possible formats. In the first format, HeLa SS6 cells arefirst transiently transfected with plasmids encoding firefly (target)and renilla (control) luciferase. DNA transfections are performed usingLipofectamine 2000 (Invitrogen) and the plasmids gWiz-Luc (Aldevron,Fargo, N. Dak.) (200 ng/well) and pRL-CMV (Promega, Madison, Wis.) (200ng/well). After 2 h, the plasmid transfection medium is removed, and thefirefly luciferase targeting siRNAs are added to the cells at variousconcentrations. In the second format, HeLa Dual-luc cells (stablyexpressing both firefly and renilla luciferase) are directly transfectedwith firefly luciferase targeting siRNAs. SiRNA transfections areperformed using either TransIT-TKO (Mirus, Madison, Wis.) orLipofectamine 2000 according to manufacturer protocols. After 24 h,cells are analyzed for both firefly and renilla luciferase expressionusing a plate luminometer (VICTOR², PerkinElmer, Boston, Mass.) and theDual-Glo Luciferase Assay kit (Promega). Firefly/renilla luciferaseexpression ratios are used to determine percent gene silencing relativeto mock-treated (no siRNA) controls.

Example 5 Serum Stability of Ligand Conjugated siRNAs

Assay:

siRNA duplexes are prepared at a stock concentration of 1 μM in whicheither the sense (S) or antisense strand (AS) contains a trace amount of5′-³²P labeled material (e.g. ³²P—S/AS and S/³²P-AS). The presence ofthe end-labeled sense or antisense strand allows for monitoring of theindividual strand within the context of the siRNA duplex. Therefore, twoduplex preparations are made for each siRNA sequence tested. siRNAduplexes are incubated in 90% human serum at a final concentration of100 nM duplex. Briefly, 2 μL of 1 μM siRNA duplex is mixed with 18 μL of100% off the clot human serum at 37° C. For a typical time course, 2 μLaliquots are removed at 10 seconds, 15 minutes, 30 minutes, 1 hour, 2hours and 4 hours and immediately quenched in 18 mL of a stop mixcontaining 90% formamide, 50 mM EDTA, 10M DTT and the dyes xylene cyanoland bromophenol blue. Samples are separated on a denaturingpolyacrylamide gel along with a control sample (4 hour buffer-aloneincubation) and a partial alkaline hydrolysis ladder used as a marker.The gel is exposed to a Fuji image plate which allows for detection ofradiolabeled siRNA and its degradation fragments.

Example 6 Binding Affinity Analysis of siRNA Containing C5-SteroiodConjugated Pyrimidines to Plasma Proteins

Method 1

Measurement of Binding Affinity:

To measure binding affinity of siRNAs to plasma protein, the 5′ end ofthe sense strand of an siRNA duplex is labeled with ³²P using T4polynucleotide kinase using standard procedures. Each of the siRNAduplexes shown in Table I will be tested in this assay. Theunincorporated label is removed using a G25 column and labeling isconfirmed by polyacrylamide gel electrophoresis. A fixed concentrationof labeled RNA (50 nM) and complementary strand (50 nM) is incubatedwith increasing concentration of plasma proteins at 25° C. for one hourin phosphate-buffered saline buffer containing 0.1 mM EDTA and 0.005%Tween 80. After incubation, the samples are loaded onto low binding,regenerated cellulose filter membranes with a molecular weight cut-offof 30,000 (Millipore). The samples are spun gently in a microfuge(NYCentrifuge 5415C; Eppendorf, Westbury, N.Y.) at 3000 rpm (735 g) for3 to 6 minutes, allowing collection of ˜20% of the loaded volume in thefiltrate.

Radioactivity present in aliquots from the filtrate and the initial(unfiltered) solutions is measured using a scintillation counter (modelLS6000IC, Beckman, Fullerton, Calif.). The counts obtained in thefiltrate aliquots represent the free (unbound) RNA, and appropriatecalculations are performed to obtain the concentration of free RNA.Further calculations yield the concentration of RNA bound to protein.See R. Zini, J. Barre, F. Bree, J. P. Tillement, B. Sebille, J.Chromatogr. 1981, 216, 191 and A. N. Kuznetsov, G. V. Gyul'khandanyan,B. Ebert, Mol. Biol. (Moscow) 1977, 11, 1057.

The extent of siRNA binding to plasma proteins is determined using anequilibrium filtration method. The fraction of bound RNA is plotted vs.the total protein concentration. The equilibrium constant, K_(d), isdetermined from nonlinear regression analysis of the fraction of siRNAbound (f_(bound)) as a function of the free protein concentration(f_(free)). Thus, the data can be fit to a two-state model:

${O + A}\overset{K_{A}}{\leftrightarrow}({OA})$where O is the unbound siRNA, A is the unbound protein, OA is thesiRNA-protein complex and K_(A) is the equilibrium association constant.Oligonucleotides to be tested are listed Example 4.Method 2Inhibition of mRNA Expression in Balb-C Mouse Treated with siRNAs:

Female BALB/c mice (6 weeks old, Harlan Sprague Dawley, Indianapolis,Ind.) are housed three to a cage under conditions meeting NationalInstitue of Health regulations. siRNAs, including unconjugated andscrambled controls (Table I, siRNAs 101-140), and vehicle containing nosiRNA are administered in 0.9% NaCl, i. p. at indicated dose levels oncedaily for three days and tissues are harvested for analysis.

Total mRNA is extracted from mouse liver by rapid homogenization of thetissue in 4 M guanidinuim isothiocyanate followed by centrifugation overa cesium chloride gradient. RNAs (20-40 μg) are resolved in 1.2% agarosegels containing 1.1% formaldehyde and transferred to nylon membranes.The blots are hybridized with a radiolabelled human cDNA probe asdescribed. Probes hybridized to mRNA transcripts are visualized andquantified using a PhosPhorImager (Molecular Dynamics). After strippingthe blots of radiolabelled probe, they are reprobed with G3PDH cDNA toconfirm equal loading.

Method 3

siRNA Treatment of Human Tumor Cells in Nude Mice—IntraperitonealInjection:

Human lung carcinoma A549 cells are harvested and 5×10⁶ cells (200 μL)were injected subcutaneously into the inner thigh of nude mice. Palpabletumors develop in approximately one month. siRNAs that target the c-rafand the H-ras messages, including steroid/lipid-conjugated RNA andscrambled controls (Table I, siRNAs 127-140) and vehicle containing nosiRNA are administered to mice intraperitoneally at a dosage of 20 mg/kgbody weight, every other day for approximately ten weeks. Mice aremonitored for tumor growth during this time.

Method 4

siRNA Treatment of Human Breast Tumor Cells in Nude Mice:

Human breast carcinoma MDA-MB-231 cells are harvested and 5×10⁵ cells(200 μL) are injected subcutaneously into the mammary fat pads ofathymic nude mice. Palpable tumors develop in approximately one month.siRNAs that target the c-raf and the H-ras messages, includingsteroid/lipid-conjugated siRNA and scrambled controls (Table I 127-140)and vehicle containing no siRNA are administered to miceintraperitoneally at a dosages of 5, 10, and 25 mg/kg/day body weight,every day for approximately 20 days. Mice are monitored for tumor growthduring this time.

Method 5

siRNA Treatment of Human Lung Tumor Cells in Nude Mice:

Human lung carcinoma A549 cells are harvested and 5×10⁶ cells (200 μL)are injected subcutaneously into the inner thigh of nude mice. Palpabletumors develop in approximately one month. siRNAs that target the c-rafand the H-ras messages, including cholesterol or cholanicacid—conjugated RNA and scrambled controls (Table I 127-140) and vehiclecontaining no siRNA are administered to mice subcutaneously at the tumorsite. Drug treatment begins one week following tumor cell inoculationand is given twice a week for four weeks. Mice are monitored for tumorgrowth for a total of nine weeks.

Method 6

Inhibition of Apo-B mRNA Expression in Hep G2 Cells and in Balb-C MouseTreated with siRNAs:

Inhibition of Aop-B mRNA expression by siRNA (Table I, siRNAs 141-146)will be evaluated in vitro and in vivo. Effect of siRNA treatment onmessage levels in HEP-G2 cells is analyzed following treatment.(following the procedure Yao, Z. Q.; Zhou, Y. X.; Guo, J.; Feng, Z. H.;Feng, X. M.; Chen, C. X.; Jiao, J. Z.; Wang, S. Q. Acta Virol. 1996February; 40(1):35-9. “Inhibition of hepatitis B virus in vitro byantisense oligonucleotides.”)

Female BALB/c mice (6 weeks old, Harlan Sprague Dawley, Indianapolis,Ind.) are housed three to a cage under conditions meeting NationalInstitue of Health regulations. siRNAs, including unconjugated andscrambled controls (Table I, siRNAs 141-146), and vehicle containing nosiRNA are administered in 0.9% NaCl, i. p. at indicated dose levels oncedaily for three days and tissues are harvested for analysis.

Total mRNA is extracted from mouse liver by rapid homogenization of thetissue in 4 M guanidinuim isothiocyanate followed by centrifugation overa cesium chloride gradient. RNAs (20-40 μg) are resolved in 1.2% agarosegels containing 1.1% formaldehyde and transferred to nylon membranes.The blots are hybridized with a radiolabelled human Apo-B cDNA probe asdescribed. Probes hybridized to mRNA transcripts are visualized andquantified using a PhosPhorImager (Molecular Dynamics). After strippingthe blots of radiolabelled probe, they are reprobed with G3PDH cDNA toconfirm equal loading.

Example 7

Table II (below) shows dU cholanic acid (L₁₇, Conjugation of 52b) and dUcholesterol (L₁₉, Conjugation of 52a) conjugated siRNAs. Therein “d”indicates a deoxy; “s” indicates a phosphorothioate linkage; and thesubscript “o” indicates indictates 2′-OMe.

TABLE II dU cholanic acid (L₁₇, Conjugation of 52b) and dU cholesterol(L₁₉, Conjugation of 52a) conjugated siRNAs (SEQ ID NOS:94-103,respectively, in order of appearance) Tar- Seq. Mass get No. Seq. Calc.Found Purity Luc 1000 ^(5′)CUUACGCUGAGUAC 6606.0 6606.45 99.2UUCGAdTdT^(3′) Luc 1001 ^(3′)dTdTGAAUGCGACU 6693.3 6693.0 89.0CAUGAAGCU^(5′) Luc 2759 ^(3′)L₁₉dTGAAUGCGACU 7259.52 7259.24 78.3CAUGAAGCU^(5′) Luc 2758 ^(5′)CUUACGCUGAGUAC 7173.41 7173.43 99.8UUCGAdTL₁₉ ^(3′) Luc 2759 ^(3′)L₁₉dTGAAUGCGACU 7259.52 7259.24 78.3CAUGAAGCU^(5′) Luc 2750 ^(5′)CUUACGCUGAGUAC 7274.5 7274.8 85.0UUCGAdTL₁₇ ^(3′) Luc 2751 ^(3′)L₁₉dTGAAUGCGACU 7189.30 7189.03 85.2CAUGAAGCU^(5′) VEGF 2701 ^(5′)GsCGGAUC₀AAACC 7205.7 7205.4 89.0UC₀ACC₀AAdTsL₁₇ ^(3′) VEGF 2702 ^(5′)GsCGGAUC₀AAACG 7219.8 7219.8 88.2UC₀AC₀C₀AAdTsL₁₇ ^(3′) VEGF 2696 ^(5′)US^(Me)U_(f)GG^(Me)U_(f)GAGG7276.3 7274.9 71.3 U^(Me)U_(F) ^(Me)U_(f)GAUCCGCd TsL₁₇ ^(3′)

Example 8

Table III (below) shows siRNA silencing of HeLa Dual Luc Cell Line (dUCholesterol). Therein “L19” indicates dU Cholesterol (obtained viasubstitution of a dT CPG support with 52a); “d” indicates a deoxy; “s”indicates a phosphorothioate linkage; and the subscript “o” indicatesindictates 2′-OMe. For IC₅₀ “a” is for “Silencing by transfected siRNAin stable HeLa Dual Luc cell line”; “b” is for “Silencing by free uptakeof siRNA in transiently transfected HeLa SS6 cell line”; and “c” is for“Silencing by free uptake of siRNA in stable HeLa dual Luc cell line”.In addition, “na” indicates no activity; and “nd” indicates notdetermined. IC₅₀ were calculated as in Example 4.

TABLE III siRNA silencing of HeLa Dual Luc Cell Line (dU Cholesterol)(SEQ ID NOS: 94-95 and 104-109, respectively, in order of appearance)Tar- Du- IC₅₀(nM) get plex Seq. Sequence a b c Luc 1 1000^(5′)CUUACGCUGAGUACUUCG 0.20 >2000 nd AdTdT^(3′) 1001^(3′)dTdTGAAUGCGACUCAUG AAGCU^(5′) Luc 2 2758 ^(5′)CUUACGCUGAGUACUUCG1.00  50.00 nd AdTL₁₉ ^(3′) 1001 ^(3′)dTGAAUGCGACUCAUGAA GCU^(5′) Luc 31000 ^(5′)CUUACGCUGAGUACUUCG 0.60  90.00 na AdTdT^(3′) 2759^(3′)L₁₉dTGAAUGCGACUCAU GAAGCU^(5′) Luc 4 2758 ^(5′)CUUACGCUGAGUACUUCG16.00 630.00  nd AdTL₁₉ ^(3′) 2759 ^(3′)L₁₉dTGAAUGCGACUCAU GAAGCU^(5′)

Example 9

Table IV and V (below) show siRNA silencing of HeLa Dual Luc Cell Line.Therein “L₁₇” indicates dU Cholanic acid (obtained via substitution of adT CPG support with 52b); “d” indicates a deoxy; subscript “F” indicatesa 2′-fluor sugar; “^(me)U” indicates a 5-methyl-uridine; “s” indicates aphosphorothioate linkage; and the subscript “o” indicates indictates2′-OMe. For IC₅₀ “a” is for “Silencing by transfected siRNA in stableHeLa Dual Luc cell line”; “b” is for “Silencing by free uptake of siRNAin transiently transfected HeLa SS6 cell line”; and “c” is for“Silencing by free uptake of siRNA in stable HeLa dual Luc cell line”.In addition, “na” indicates no activity; and “nd” indicates notdetermined. IC₅₀ were calculated as in Example 4.

In Table V the efficacy is set at “+++” for the control (unmodified)duplex 9 and the other duplexes (10-12) are rated accordingly.

TABLE IV siRNA silencing of HeLa Dual Luc Cell Line (SEQ ID NOS: 94-95and 110-115, respectively, in order of appearance) Tar- Du- IC₅₀(nM) getplex Seq. Sequence a b c Luc 5 1000 ^(5′)CUUACGCUGAGUACUUCG 0.20 >2000nd AdTdT^(3′) 1001 ^(3′)dTdTGAAUGCGACUCAUG AAGCU^(5′) Luc 6 2750^(5′)CUUACGCUGAGUAGUUCG 0.03 110.00 nd AdTL₁₇ ^(3′) 1001^(3′)dTGAAUGCGACUCAUGAA GCU^(5′) Luc 7 1000 ^(5′)CUUACGCUGAGUACUUGG 0.04200.00 nd AdTdT^(3′) 2759 ^(3′)L₁₇dTGAAUGCGACUCAU GAAGGU^(5′) Luc 8 2758^(5′)CUUACGCUGAGUACUUCG 0.70  50.00 na AdTL₁₇ ^(3′) 2759^(3′)L₁₇dTGAAUGCGACUCAU GAAGCU^(5′)

TABLE V siRNA silencing of HeLa Dual Luc Cell Line (SEQ ID NOS: 116-123,respectively, in order of appearance) Tar- Dup- Effi- get lex Seq.Sequence cacy VEGF  9 4112 GCGGAUCAAACCUCACCAAdTdT +++ 4180dTdTCGCCUAGUUUGGAGUGGUU VEGF 10 2701 GsCGGAUC₀AAACCUC₀ACC₀AAdTsL₁₇ ++2381 UsUGGUGAGGUUUGAUCCGCdTsdT VEGF 11 2702GsCGGAUC₀AAACCUC₀AC₀C₀AAdTsL₁₇ ++ 2381 UsUGGUGAGGUUUGAUCCGCdTsdT VEGF 124112 GCGGAUCAAACCUCACCAATT +++ 2696Us^(5Me)U_(F)G^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GA UCCGCdTsL₁₇

TABLE IV siRNA silencing of HeLa Dual Luc Cell Line (SEQ ID NOS: 94-95and 110-115, respectively, in order of appearance) Tar- Du- IC₅₀(nM) getplex Seq. Sequence a b c Luc 5 1000 ^(5′)CUUACGCUGAGUACUUCG 0.20 >2000nd AdTdT^(3′) 1001 ^(3′)dTdTGAAUGCGACUCAUG AAGCU^(5′) Luc 6 2750^(5′)CUUACGCUGAGUAGUUCG 0.03 110.00 nd AdTL₁₇ ^(3′) 1001^(3′)dTGAAUGCGACUCAUGAA GCU^(5′) Luc 7 1000 ^(5′)CUUACGCUGAGUACUUGG 0.04200.00 nd AdTdT^(3′) 2759 ^(3′)L₁₇dTGAAUGCGACUCAU GAAGGU^(5′) Luc 8 2758^(5′)CUUACGCUGAGUACUUCG 0.70  50.00 na AdTL₁₇ ^(3′) 2759^(3′)L₁₇dTGAAUGCGACUCAU GAAGCU^(5′)

TABLE V siRNA silencing of HeLa Dual Luc Cell Line (SEQ ID NOS: 116-123,respectively, in order of appearance) Tar- Dup- Effi- get lex Seq.Sequence cacy VEGF  9 4112 GCGGAUCAAACCUCACCAAdTdT +++ 4180dTdTCGCCUAGUUUGGAGUGGUU VEGF 10 2701 GsCGGAUC₀AAACCUC₀ACC₀AAdTsL₁₇ ++2381 UsUGGUGAGGUUUGAUCCGCdTsdT VEGF 11 2702GsCGGAUC₀AAACCUC₀AC₀C₀AAdTsL₁₇ ++ 2381 UsUGGUGAGGUUUGAUCCGCdTsdT VEGF 124112 GCGGAUCAAACCUCACCAATT 2696Us^(5Me)U_(F)G^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GA +++ UCCGCdTsL₁₇

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A single-stranded siRNA or microRNA represented by formula I:

wherein X is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂, or—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂; M represents independently for eachoccurrence an alkali metal or a transition metal with an overall chargeof +1; n is 16, 17, 18, 19, 20, 21, 22, 23, or 24; R¹, R², and R⁵represent independently for each occurrence H, OH, F, —Oalkyl, —Oallyl,—O(C(R⁷)₂)_(v)OR⁷, —O(C(R⁷)₂)_(v)SR⁷, —O(C(R⁷)₂)_(v)N(R⁷)₂,—O(C(R⁷)₂)_(v)C(O)N(R⁷)₂, —N(R⁷)₂, —S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C₁-C₆)alkyl, —O(C(R⁷)₂)_(v)S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C(R⁷)₂)_(v)N((C₁-C₆)alkyl)₂, or—O(C(R⁷)₂)_(v)ON((C₁-C₆)alkyl)₂; A¹ represents independently for eachoccurrence:

Z¹ represents independently for each occurrence O or S; Z² representsindependently for each occurrence —OH, —OM, —Oalkyl, —Oaryl, —Oaralkyl,—SH, SM, —Salkyl, —Saryl, —Saralkyl, —N(R³)R⁴, —(C(R⁷)₂)_(m)N(R⁷)₂,—N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or alkyl; R³ and R⁴ are H or alkyl; or R³ andR⁴ taken together form a 3-, 4-, 5-, 6-, or 7-member ring; R⁶ representsindependently for each occurrence H, alkyl, or —NHCH₂CH═CH₂; R⁷represents independently for each occurrence H or alkyl; A² representsindependently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence the formula:

B¹ represents independently for each occurrence a bond between A³ andA⁴; B² represents independently for each occurrence a bond between A⁴and A⁵; R represents independently for each occurrence hydrogen oralkyl; m represents independently for each occurrence 1, 2, 3, 4, 5, 6,7 or 8; m¹ represents independently for each occurrence 0, 1, 2, 3, 4,5, 6, 7 or 8; v represents independently for each occurrence 1, 2, 3, or4; w represents independently for each occurrence 1, 2, or 3 in accordwith the rules of valence; provided that A⁵ occurs at least once; and A⁵represents independently for each occurrence compound C, compound C′,compound D, compound D′ or a radical of a steroid, bile acid, lipid,folic acid, pyridoxal, B12, riboflavin, aromatic compound, polycycliccompound, crown ether, intercalator, cleaver molecule, protein-bindingagent, carbohydrate, or an optionally substituted saturated 5-memberedring; wherein compound C is represented by:

and compound C′ is represented by:

wherein for C and C′ R²⁵ represents independently for each occurrence H,halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R²⁷)₂, —CO₂R²⁷,—COR²⁷, —OC(O)R²⁷, alkyl, alkenyl, aryl, or aralkyl; R²⁶ representsindependently for each occurrence alkyl alkenyl, aryl, or aralkyl; R²⁷represents independently for each occurrence H, alkyl, alkenyl, aryl, oraralkyl; R²⁸ represents independently for each occurrence —OC(O)R²⁷,—N(R²⁷)C(O)R²⁷, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, alkyl, alkenyl, aryl, oraralkyl; and p represents independently for each occurrence 1, 2, 3, 4,5, 6, or 7; and compound D is represented by:

and compound D′ is represented by:

wherein for D and D′ R²⁹ represents independently for each occurrence H,halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R³⁰)₂, —CO₂R³⁰,—COR³⁰, —OC(O)R³⁰, alkyl, alkenyl, aryl, or aralkyl; R³⁰ representsindependently for each occurrence H, alkyl, alkenyl, aryl, or aralkyl;R³¹ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl; and t represents independently for each occurrence 1, 2, 3,4, 5, 6, or
 7. 2. The single-stranded siRNA or microRNA of claim 1,wherein n is 18, 19, 20, 21, or
 22. 3. The single-stranded siRNA ormicroRNA of claim 1, wherein n is
 20. 4. The single-stranded siRNA ormicroRNA of claim 1, wherein A⁵ occurs at least twice.
 5. Thesingle-stranded siRNA or microRNA of claim 1, wherein A⁵ representsindependently for each occurrence compound C, compound D, or a radicalof cholesterol, 5β-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fuisidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano. 6.The single-stranded siRNA or microRNA of claim 1, wherein A⁵ representsindependently for each occurrence a radical of cholesterol or5β-cholanic acid.
 7. The single-stranded siRNA or microRNA of claim 1,wherein A⁵ represents independently for each occurrence:

wherein R²⁰ represents independently for each occurrence hydroxyl,amino, halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, oracyloxy; Z³ represents independently for each occurrence O, S, or NR;and y represents independently for each occurrence 0, 1, 2, 3, 4, 5, or6 in accord with the rules of valence.
 8. A double-stranded siRNA ormicroRNA comprising a first strand and a second strand, wherein saidfirst strand and said second strand are represented independently byformula II:

X is H, —P(O)(OM)₂, —P(O)(OM)-O—P(O)(OM)₂, —P(O)(Oalkyl)₂, or—P(O)(Oalkyl)-O—P(O)(Oalkyl)₂; M represents independently for eachoccurrence an alkali metal or a transition metal with an overall chargeof +1; n is 16, 17, 18, 19, 20, 21, 22, 23, or 24; R¹, R², and R⁵represent independently for each occurrence H, OH, F, —Oalkyl, —Oallyl,—O(C(R⁷)₂)_(v)OR⁷, —O(C(7)₂)_(v)SR⁷, —O(C(R⁷)₂)_(v)N(R⁷)₂,—O(C(R⁷)₂)_(v)C(O)N(R⁷)₂, —N(R⁷)₂, —S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C₁-C₆)alkyl, —O(C(R⁷)₂)_(v)S(C₁-C₆)alkyl,—O(C(R⁷)₂)_(v)O(C(R⁷)₂)_(v)N((C₁-C₆)alkyl)₂, or—O(C(R⁷)₂)_(v)ON((C₁-C₆)alkyl)₂; A¹ represents independently for eachoccurrence:

Z¹ represents independently for each occurrence O or S; Z² representsindependently for each occurrence —OH, —OM, —Oalkyl, —Oaryl, —Oaralkyl,—SH, SM, —Salkyl, —Saryl, —Saralkyl, —N(R³)R⁴, —(C(R⁷)₂)_(m)N(R⁷)₂,—N(R⁷)(C(R⁷)₂)_(m)N(R⁷)₂, or alkyl; R³ and R⁴ are H or alkyl; or R³ andR⁴ taken together form a 3-, 4-, 5-, 6-, or 7-member ring; R⁶ representsindependently for each occurrence H, alkyl, or —NHCH₂CH═CH₂; R⁷represents independently for each occurrence H or alkyl; A² representsindependently for each occurrence:

A³ represents independently for each occurrence

A⁴ represents independently for each occurrence the formula:

B¹ represents independently for each occurrence a bond between A³ andA⁴; B² represents independently for each occurrence a bond between A⁴and A⁵; R represents independently for each occurrence hydrogen oralkyl; m represents independently for each occurrence 1, 2, 3, 4, 5, 6,7 or 8; m¹ represents independently for each occurrence 0, 1, 2, 3, 4,5, 6, 7 or 8; v represents independently for each occurrence 1, 2, 3, or4; w represents independently for each occurrence 1, 2, or 3 in accordwith the rules of valence; provided that A⁵ occurs at least once; and A⁵represents independently for each occurrence compound C, compound D, ora radical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12,riboflavin, biotin, aromatic compound, polycyclic compound, crown ether,intercalator, cleaver molecule, protein-binding agent, carbohydrate, oran optionally substituted saturated 5-membered ring; wherein compound Cis represented by:

and compound C′ is represented by:

wherein for C and C′ R²⁵ represents independently for each occurrence H,halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R²⁷)₂, —CO₂R²⁷,—COR²⁷, —OC(O)R²⁷, alkyl, alkenyl, aryl, or aralkyl; R²⁶ representsindependently for each occurrence alkyl, alkenyl, aryl, or aralkyl; R²⁷represents independently for each occurrence H, alkyl, alkenyl, aryl, oraralkyl; R²⁸ represents independently for each occurrence —OC(O)R²⁷,—N(R²⁷)C(O)R²⁷, —N(R²⁷)₂, —CO₂R²⁷, —COR²⁷, alkyl, alkenyl, aryl, oraralkyl; and p represents independently for each occurrence 1, 2, 3, 4,5, 6, or 7; and compound D is represented by:

and compound D′ is represented by:

wherein for D and D′ R²⁹ represents independently for each occurrence H,halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R³⁰)₂, —CO₂R³⁰,—COR³⁰, —OC(O)R³⁰, alkyl, alkenyl, aryl, or aralkyl; R³⁰ representsindependently for each occurrence H, alkyl, alkenyl, aryl, or aralkyl;R³¹ represents independently for each occurrence alkyl, alkenyl, aryl,or aralkyl; and t represents independently for each occurrence 1, 2, 3,4, 5, 6, or
 7. 9. The double-stranded siRNA of claim 8, wherein n is 18,19, 20, 21, or
 22. 10. The double-stranded siRNA of claim 8, wherein nis
 20. 11. The double-stranded siRNA of claim 8, wherein n is 20, andsaid first strand and said second strand are hybridized so that thereare two unhybridized nucleotides on said first strand and said secondstrand.
 12. The double-stranded siRNA of claim 8, wherein n is 20 forsaid first strand, and n is 22 for said second strand.
 13. Thedouble-stranded siRNA of claim 8, wherein A⁵ occurs at least twice. 14.The double-stranded siRNA of claim 8, wherein A⁵ occurs only in saidfirst strand.
 15. The double-stranded siRNA of claim 8, wherein saidfirst strand and said second strand each contain at least one occurrenceof A⁵.
 16. The double-stranded siRNA of claim 8, wherein A⁵ representsindependently for each occurrence compound C, compound D, or a radicalof cholesterol, 5β-cholanic acid, progesterone, aldosterone,dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol,dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone,testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol,corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon,equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol,mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene,fusidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol,dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone,hydrocortisone, parametasone, betametasone, cortisone, fluocinonide,fluorometholone, halcinonide, budesonide, or any one of them furthersubstituted with one or more of hydroxyl, halogen, amino, alkylamino,alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano. 17.The double-stranded siRNA of claim 8, wherein A⁵ representsindependently for each occurrence a radical of cholesterol or5β-cholanic acid.
 18. The double-stranded siRNA of claim 8, wherein A⁵represents independently for each occurrence:

wherein R²⁰ represents independently for each occurrence hydroxyl,amino, halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, oracyloxy; Z³ represents independently for each occurrence O, S, or NR;and y represents independently for each occurrence 0, 1, 2, 3, 4, 5, or6 in accord with the rules of valence.
 19. The single-stranded siRNA ormicroRNA of claim 1, wherein A⁴ independently represents for eachoccurrence the formula:


20. The single-stranded siRNA or microRNA of claim 19, wherein A⁴independently represents for each occurrence the formula:


21. The single-stranded siRNA or microRNA of claim 5, wherein A⁴independently represents for each occurrence the formula:


22. The single-stranded siRNA or microRNA of claim 21, wherein A⁴independently represents for each occurrence the formula:


23. The single-stranded siRNA or microRNA of claim 6, wherein A⁴independently represents for each occurrence the formula:


24. The single-stranded siRNA or microRNA of claim 23, wherein A⁴independently represents for each occurrence the formula:


25. The single-stranded siRNA or microRNA of claim 7, wherein A⁴independently represents for each occurrence the formula:


26. The single-stranded siRNA or microRNA of claim 25, wherein A⁴independently represents for each occurrence the formula:


27. The single-stranded siRNA of claim 8, wherein A⁴ independentlyrepresents for each occurrence the formula:


28. The single-stranded siRNA of claim 27, wherein A⁴ independentlyrepresents for each occurrence the formula:


29. The single-stranded siRNA of claim 16, wherein A⁴ independentlyrepresents for each occurrence the formula:


30. The single-stranded siRNA of claim 29, wherein A⁴ independentlyrepresents for each occurrence the formula:


31. The single-stranded siRNA of claim 17, wherein A⁴ independentlyrepresents for each occurrence the formula:


32. The single-stranded siRNA of claim 31, wherein A⁴ independentlyrepresents for each occurrence the formula:


33. The single-stranded siRNA of claim 18, wherein A⁴ independentlyrepresents for each occurrence the formula:


34. The single-stranded siRNA of claim 33, wherein A⁴ independentlyrepresents for each occurrence the formula: